Methods for Treatment of Alport Syndrome

ABSTRACT

Provided herein are methods for the treatment of Alport Syndrome, using modified oligonucleotides targeted to miR-21. In certain embodiments, a modified oligonucleotide targeted to miR-21 improves kidney function and/or reduces fibrosis in subjects having Alport Syndrome. In certain embodiments, administration of a modified oligonucleotide targeted to miR-21 delays the onset of end-stage renal disease in a subject having Alport Syndrome. In certain embodiments, a modified oligonucleotide targeted to miR-21 delays the need for dialysis or kidney transplant in a subject having Alport Syndrome.

This application is a continuation of U.S. application Ser. No.16/747,837, filed Jan. 21, 2020, which is a continuation of U.S.application Ser. No. 16/655,085, filed Oct. 16, 2019, now abandoned,which is a continuation of U.S. application Ser. No. 16/352,324, filedMar. 13, 2019, which is a continuation of U.S. application Ser. No.15/948,450, filed Apr. 9, 2018, now abandoned, which is a continuationof U.S. application Ser. No. 15/606,554, filed May 26, 2017, now U.S.Pat. No. 9,970,011, which is a continuation of U.S. application Ser. No.15/151,290, filed May 10, 2016, now U.S. Pat. No. 9,688,986, which is acontinuation of U.S. application Ser. No. 14/677,387, filed Apr. 2,2015, now U.S. Pat. No. 9,359,609, which is a continuation of U.S.application Ser. No. 14/048,827, filed Oct. 8, 2013, now U.S. Pat. No.9,012,423, which claims the benefit of U.S. Provisional Application No.61/711,514, filed Oct. 9, 2012, and U.S. Provisional Application No.61/779,137, filed Mar. 13, 2013, each of which is incorporated byreference herein in its entirety for any purpose.

FIELD OF INVENTION

Provided herein are methods and compositions for the treatment of AlportSyndrome.

DESCRIPTION OF RELATED ART

Type IV collagen, a major component of the basement membrane, is afamily of six alpha chains: alpha-1 collagen (Type IV), alpha-2 collagen(Type IV), alpha-3 collagen (Type IV), alpha-4 collagen (Type IV),alpha-5 collagen (Type IV), and alpha-6 collagen (Type IV). The alpha-3,alpha-4 and alpha-6 chains of collagen IV are fundamental components ofthe collagen network of the glomerular basement membrane (GBM), whichperforms the critical function of filtration of blood by the kidney.

Alport Syndrome is an inherited form of kidney disease in which anabnormal type of glomerular basement membrane (GBM) is produced, leadingto interstitial fibrosis, glomerular sclerosis and eventual loss ofkidney function. The disease is also frequently characterized by hearingdefects and ocular anomalies. Alport Syndrome is caused by a mutation inCol4a3, Col4a4, or Col4a5, which encode the alpha3(IV), alpha4(IV), andalpha5(IV) chains of type IV collagen, respectively. Mutations in theCol4a5 gene on the X chromosome cause the X-linked form of AlportSyndrome, which accounts for 85% of all cases of the disease. Anautosomal recessive form is due to inheritance of mutations in each copyof either Col4a3 or Col4a4, each of which is located on chromosome 2.The rare autosomal dominant form is due to inheritance of adominant-negative mutation in either the Col4a3 or Col4a4 gene. TheX-linked form is more severe in males than in females, with most casesin males progressing to end-stage renal disease (ESRD). The autosomalform is of similar severity in males and females. Most cases of thedisease are due to an inherited mutation, but some cases are due to a denovo mutation in one of the Col4aA genes.

SUMMARY OF INVENTION

Provided here are methods for treating Alport Syndrome comprisingadministering to a subject having or suspected of having Alport Syndromea modified oligonucleotide consisting of 12 to 25 linked nucleosides,wherein the nucleobase sequence of the modified oligonucleotide iscomplementary to miR-21. In certain embodiments, the subject has beendiagnosed as having Alport Syndrome prior to administering the modifiedoligonucleotide. In certain embodiments, the subject, prior toadministration of the modified oligonucleotide, was determined to havean increased level of miR-21 in the kidney tissue of the subject. Incertain embodiments, the subject, prior to administration of themodified oligonucleotide, was determined to have an increased level ofmiR-21 in the urine or blood of the subject.

In any of the embodiments provided herein, administration of a modifiedoligonucleotide complementary to miR-21, to a subject having orsuspected of having Alport Syndrome, may improve kidney function; delaythe onset of end stage renal disease; delay time to dialysis; delay timeto renal transplant; and/or improve life expectancy in the subject.

In any of the embodiments provided herein, administration of a modifiedoligonucleotide complementary to miR-21, to a subject having orsuspected of having Alport Syndrome may reduce hematuria; delay theonset of hematuria; reduce proteinuria; delay the onset of proteinuria;reduce kidney fibrosis; slow further progression of fibrosis; and/orhalt further progression of fibrosis.

In any of the embodiments provided herein, the subject may have amutation selected from a mutation in the gene encoding the alpha 3 chainof type IV collagen, a mutation in the gene encoding the alpha 4 chainof type IV collagen, or a mutation in the gene encoding the alpha 5chain of type IV collagen. In certain embodiments, the subject is male.In certain embodiments, the subject is female. In certain embodiments,the subject is identified as having hematuria, and/or proteinuria. Incertain embodiments, the subject has reduced kidney function. In certainembodiments, the subject is in need of improved kidney function.

Any of the embodiments provided herein may comprise measuring blood ureanitrogen in the blood of the subject; measuring creatinine in the bloodof the subject; measuring creatinine clearance in the subject; measuringproteinuria in the subject; measuring albumin:creatinine ratio in thesubject; and/or measuring glomerular filtration rate in the subject.

Any of the embodiments provided herein may comprise measuringN-acetyl-β-D-glucosaminidase (NAG) protein in the urine of the subject;measuring neutrophil gelatinase-associated lipocalin (NGAL) protein inthe urine of the subject; measuring kidney injury molecule-1 (KIM-1)protein in the urine of the subject; measuring interleukin-18 (IL-18)protein in the urine of the subject; measuring monocyte chemoattractantprotein (MCP1) levels in the urine of the subject; measuring connectivetissue growth factor (CTGF) levels in the urine of the subject;measuring collagen IV (Col IV) fragments in the urine of the subject;measuring collagen III (Col III) fragments in the urine of the subject;and/or measuring podocyte protein levels in the urine of the subject,wherein the podocyte protein is selected from nephrin and podocin. Anyof the embodiments provided herein may comprise measuring cystatin Cprotein in the blood of a subject; measuring β-trace protein (BTP) inthe blood of a subject; and measuring 2-microglobulin (B2M) in the bloodof a subject.

Any of the methods provided herein may improve one or more markers ofkidney function in the subject, selected from reduced blood ureanitrogen in the subject; reduced creatinine in the blood of the subject;improved creatinine clearance in the subject; reduced proteinuria in thesubject; reduced albumin:creatinine ratio in the subject; and/orimproved glomerular filtration rate in the subject. Any of the methodsprovided herein may improve one or more markers of kidney function inthe subject, selected from reduced NAG in the urine of the subject;reduced NGAL in the urine of the subject; reduced KIM-1 in the urine ofthe subject; reduced IL-18 in the urine of the subject; reduced MCP1 inthe urine of the subject; reduced CTGF in the urine of the subject;reduced collagen IV fragments in the urine of the subject; reducedcollagen III fragments in the urine of the subject; and reduced podocyteprotein levels in the urine of the subject, wherein the podocyte proteinis selected from nephrin and podocin. Any of the methods provided hereinmay improve one or more markers of kidney function selected from reducedcystatin C protein in the blood of a subject, reduced β-trace protein(BTP) in the blood of a subject, and reduced 2-microglobulin (B2M) inthe blood of a subject.

In any of the embodiments provided herein, the proteinuria isalbuminuria. The albuminuria may be high normal albuminuria,microalbuminuria, or macroalbuminuria.

In certain embodiments, the Alport Syndrome is the X-linked form ofAlport Syndrome. In certain embodiments, the Alport Syndrome is theautosomal form of Alport Syndrome.

Any of the embodiments provided herein may comprise administering atleast one additional therapy selected from an angiotensin II convertingenzyme (ACE) inhibitor, an angiotensin II receptor blocker (ARB), ananti-hypertensive agent, a vitamin D analog, an oral phosphate binder,dialysis, and kidney transplant. In any of these embodiments, theangiotensin II converting enzyme (ACE) inhibitors is selected fromcaptopril, enalapril, lisinopril, benazepril, quinapril, fosinopril, andramipril. In any of these embodiments, the angiotensin II receptorblockers (ARB) is selected from candesartan, irbesartan, olmesartan,losartan, valsartan, telmisartan, and eprosartan. In any of theseembodiments, an ACE inhibitor is selected from cilazapril, perindopril,and trandolapril.

In certain embodiments, an ACE inhibitor is administered at a doseranging from 0.5 to 1 mg/m²/day, from 1 to 6 mg/m²/day, from 1 to 2mg/m²/day, from 2 to 4 mg/m²/day, or from 4 to 8 mg/m²/day.

In certain embodiments, an ARB is administered at a dose ranging from6.25 to 150 mg/m2/day. In any of these embodiments, an ARB isadministered at a dose of 6.25 mg/m²/day, 10 mg/m²/day, 12.5 mg/m²/day,18.75 mg/m²/day, 37.5 mg/m²/day, 50 mg/m²/day, or 150 mg/m²/day.

In certain embodiments, the at least one additional therapy is analdosterone antagonsist. In certain embodiments, an aldosteroneantagonist is spironolactone. In certain embodiments, spironolactone isadministered at a dose ranging from 10 to 35 mg daily. In certainembodiments, spironolactone is administered at a dose of 25 mg daily.

In any of the embodiments provided herein, the nucleobase sequence ofthe modified oligonucleotide is at least 90% complementary, is at least95% complementary, or is 100% complementary to the nucleobase sequenceof miR-21 (SEQ ID NO: 1).

In any of the embodiments provided herein, the modified oligonucleotideconsists of 8 to 30, 12 to 25, or 15 to 25 linked nucleosides. In any ofthe embodiments provided herein, the modified oligonucleotide consistsof 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 linked nucleosides. Inany of the embodiments provided herein, the modified oligonucleotideconsists of 15, 16, 17, 18, 19, 20, 21, or 22 linked nucleosides.

In any of the embodiments provided herein, the modified oligonucleotidecomprises at least one modified nucleoside. The modified nucleoside maybe selected from an S-cEt nucleoside, a 2′-O-methoxyethyl nucleoside,and an LNA nucleoside. The modified oligonucleotide may comprise atleast one modified internucleoside linkage. Each internucleoside linkageof the modified oligonucleotide may be a modified internucleosidelinkage. In certain embodiments, the modified internucleoside linkage isa phosphorothioate internucleoside linkage.

In any of the embodiments provided herein, the modified oligonucleotidemay have the structure5′-A_(E)C_(S)ATC_(S)AGTC_(S)TGAU_(S)AAGC_(S)TA_(E)-3′, (SEQ ID NO: 3)where nucleosides not followed by a subscript indicateβ-D-deoxyribonucleosides; nucleosides followed by a subscript “E”indicate 2′-MOE nucleosides; nucleosides followed by a subscript “S”indicate S-cEt nucleosides, and each internucleoside linkage is aphosphorothioate internucleoside linkage.

Provided herein is the use of a modified oligonucleotide consisting of 8to 30, 12 to 25, or 15 to 25 linked nucleosides, wherein the nucleobasesequence of the modified oligonucleotide is complementary to miR-21, forthe treatment of Alport Syndrome.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-B. Anti-miR-21 improves kidney function of Col4a3−/− mice. Bloodurea nitrogen (BUN) at 9 weeks (A) and urinary albumin/creatinine ratioat weeks 3, 5, 7 and 9 (B). * indicates statistical significance.

FIG. 2. Anti-miR-21 prevents glomerulosclerosis in Col4a3−/− mice.Glomerulosclerosis was evaluated using semi-quantitative sclerosisscores ranging from 0 (no sclerosis) to 4 (complete sclerosis) (n=10).

FIG. 3A-B. Anti-miR-21 reduces fibrosis in Col4a3−/− mice. (A)Quantification of Picrosirius (Sirius) red-stained fibrosis in kidney(n=6) and (B) Quantitative PCR of Col1a1 transcripts normalized to GAPDH(n=6). * indicates statistical significance.

FIG. 4A-C. Anti-miR-21 reduces kidney injury ranking in Col4a3−/− mice.(A) Kidney injury rank score of renal sections of 9 week old mice. (B)Proportion of glomerular crescents (n=5). (C) Quantification of tubuleinjury (n=5). * indicates statistical significance.

FIG. 5A-B Anti-miR-21 reduces macrophage infiltration (A) and decreasesmyofibroblasts (B) in Col4a3−/− mice. (A) Quantification of F4/80 stainof renal sections of 9 week old mice (n=5). (B) Quantification of αSMAstain of renal sections of 9 week old mice (n=5). * indicatesstatistical significance.

FIG. 6A-B. Anti-miR-21 reduces reactive oxygen species in Col4a3−/−mice. (A) Quantification of hydrogen peroxide in urine of Col4a3−/− micetreated with anti-miR-21 or PBS control (n=8; * indicates statisticalsignificance); (B) Quantification of DES in kidney tissue of Col4a3−/−mice treated with anti-miR-21 or PBS control, and in wild type mice (n=3per group; *indicates statistical significance).

FIG. 7. Anti-miR-21 improves podocyte number in Col4a3−/− mice.Quantification of number of WT1-positive cells in the glomerulus ofCol4a3−/− mice treated with anti-miR-21 or PBS control (n=3; p=0.005).

FIG. 8A-B. Anti-miR-21 increases lifespan of Col4a3−/− mice. (A) Weightof Col4a3−/− mice treated with anti-miR-21 or PBS control (p<0.01); (B)Lifespan of Col4a3−/− mice treated with anti-miR-21 or PBS control(p<0.001).

FIG. 9A-B. Anti-miR-21 improves kidney function and increases lifespanof Col4a3−/− mice in a dose-responsive manner (n=10-13 per treatmentgroup). (A) Blood urea nitrogen at 7 weeks; (B) Lifespan of Col4a3−/−mice treated with anti-miR-21 at multiple doses, once weekly (QW) ortwice weekly (BIW), or PBS control.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in thearts to which the invention belongs. Unless specific definitions areprovided, the nomenclature utilized in connection with, and theprocedures and techniques of, analytical chemistry, synthetic organicchemistry, and medicinal and pharmaceutical chemistry described hereinare those well-known and commonly used in the art. In the event thatthere is a plurality of definitions for terms herein, those in thissection prevail. Standard techniques may be used for chemical synthesis,chemical analysis, pharmaceutical preparation, formulation and delivery,and treatment of subjects. Certain such techniques and procedures may befound for example in “Carbohydrate Modifications in Antisense Research”Edited by Sangvi and Cook, American Chemical Society, Washington D.C.,1994; and “Remington's Pharmaceutical Sciences,” Mack Publishing Co.,Easton, Pa., 18th edition, 1990; and which is hereby incorporated byreference for any purpose. Where permitted, all patents, patentapplications, published applications and publications, GENBANKsequences, websites and other published materials referred to throughoutthe entire disclosure herein, unless noted otherwise, are incorporatedby reference in their entirety. Where reference is made to a URL orother such identifier or address, it is understood that such identifierscan change and particular information on the internet can change, butequivalent information can be found by searching the internet. Referencethereto evidences the availability and public dissemination of suchinformation.

Before the present compositions and methods are disclosed and described,it is to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting. It must be noted that, as used in the specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise.

Definitions

“Alport Syndrome” means an inherited form of kidney disease in which anabnormal level of glomerular basement membrane (GBM) is produced,leading to interstitial fibrosis, glomerular sclerosis and eventual lossof kidney function. The disease is also frequently characterized byhearing defects and ocular anomalies.

“Hematuria” means the presence of red blood cells in the urine.

“Albuminuria” means the presence of excess albumin in the urine, andincludes without limitation, normal albuminuria, high normalalbuminuria, microalbuminuria and macroalbuminuria. Normally, theglomerular filtration permeability barrier, which is composed ofpodocyte, glomerular basement membrane and endothelial cells, preventsserum protein from leaking into urine. Albuminuria may reflect injury ofthe glomerular filtration permeability barrier. Albuminuria may becalculated from a 24-hour urine sample, an overnight urine sample or aspot-urine sample.

“High normal albuminuria” means elevated albuminuria characterized by(i) the excretion of 15 to <30 mg of albumin into the urine per 24 hoursand/or (ii) an albumin/creatinine ratio of 1.25 to <2.5 mg/mmol (or 10to <20 mg/g) in males or 1.75 to <3.5 mg/mmol (or 15 to <30 mg/g) infemales.

“Microalbuminuria” means elevated albuminuria characterized by (i) theexcretion of 30 to 300 mg of albumin into the urine per 24 hours and/or(ii) an albumin/creatinine ratio of 2.5 to <25 mg/mmol (or 20 to <200mg/g) in males or 3.5 to <35 mg/mmol (or 30 to <300 mg/g) in females.

“Macroalbuminuria” means elevated albuminuria characterized by theexcretion of more than 300 mg of albumin into the urine per 24 hoursand/or (ii) an albumin/creatinine ratio of >25 mg/mmol (or >200 mg/g) inmales or >35 mg/mmol (or >300 mg/g) in females.

“Albumin/creatinine ratio” means the ratio of urine albumin (mg/dL) perurine creatinine (g/dL) and is expressed as mg/g. In certainembodiments, albumin/creatinine ratio may be calculated from aspot-urine sample and may be used as an estimate of albumin excretionover a 24 hour period.

“Estimated glomerular filtration rate (eGFR) or “glomerular filtrationrate (GFR)” means a measurement of how well the kidneys are filteringcreatinine, and is used as an estimate of how much blood passes throughthe glomeruli per minute. Normal results may range from 90-120mL/min/1.73 m². Levels below 60 mL/min/1.73 m² for 3 or more months maybe an indicator chronic kidney disease. Levels below 15 mL/min/1.73 m²may be an indicator of kidney failure.

“Proteinuria” means the presence of an excess of serum proteins in theurine. Proteinuria may be characterized by the excretion of >250 mg ofprotein into the urine per 24 hours and/or a urine protein to creatinineratio of ≥0.20 mg/mg. Serum proteins elevated in association withproteinuria include, without limitation, albumin.

“Blood urea nitrogen” or “BUN” means a measure of the amount of nitrogenin the blood in the form of urea. The liver produces urea in the ureacycle as a waste product of the digestion of protein, and the urea isremoved from the blood by the kidneys. Normal human adult blood maycontain between 7 to 21 mg of urea nitrogen per 100 ml (7-21 mg/dL) ofblood. Measurement of blood urea nitrogen is used as an indicator ofrenal health. If the kidneys are not able to remove urea from the bloodnormally, a subject's BUN rises.

“End stage renal disease (ESRD)” means the complete or almost completefailure of kidney function.

“Impaired kidney function” means reduced kidney function, relative tonormal kidney function.

“Fibrosis” means the formation or development of excess fibrousconnective tissue in an organ or tissue. In certain embodiments,fibrosis occurs as a reparative or reactive process. In certainembodiments, fibrosis occurs in response to damage or injury. The term“fibrosis” is to be understood as the formation or development of excessfibrous connective tissue in an organ or tissue as a reparative orreactive process, as opposed to a formation of fibrous tissue as anormal constituent of an organ or tissue.

“Slows further progression” means to reduce the rate at which a medicalcondition moves towards an advanced state.

“Halts further progression” means to stop progression of a medicalcondition to an advanced state.

“Delay time to dialysis” means to maintain sufficient kidney functionsuch that the need for dialysis treatment is delayed.

“Delay time to renal transplant” means to maintain sufficient kidneyfunction such that the need for a kidney transplant is delayed.

“Improves life expectancy” means to lengthen the life of a subject bytreating one or more symptoms of a disease in the subject.

“Anti-miR” means an oligonucleotide having a nucleobase sequencecomplementary to a microRNA. In certain embodiments, an anti-miR is amodified oligonucleotide.

“Anti-miR-X” where “miR-X” designates a particular microRNA, means anoligonucleotide having a nucleobase sequence complementary to miR-X. Incertain embodiments, an anti-miR-X is fully complementary (i.e., 100%complementary) to miR-X. In certain embodiments, an anti-miR-X is atleast 80%, at least 85%, at least 90%, or at least 95% complementary tomiR-X. In certain embodiments, an anti-miR-X is a modifiedoligonucleotide.

“miR-21” means the mature miRNA having the nucleobase sequence

(SEQ ID NO: 1) UAGCUUAUCAGACUGAUGUUGA.

“miR-21 stem-loop sequence” means the stem-loop sequence having thenucleobase sequence

(SEQ ID NO: 2) UGUCGGGUAGCUUAUCAGACUGAUGUUGACUGUUGAAUCUCAUGGCAACACCAGUCGAUGGGCUGUCUGACA.

“Target nucleic acid” means a nucleic acid to which an oligomericcompound is designed to hybridize.

“Targeting” means the process of design and selection of nucleobasesequence that will hybridize to a target nucleic acid.

“Targeted to” means having a nucleobase sequence that will allowhybridization to a target nucleic acid.

“Modulation” means a perturbation of function, amount, or activity. Incertain embodiments, modulation means an increase in function, amount,or activity. In certain embodiments, modulation means a decrease infunction, amount, or activity.

“Expression” means any functions and steps by which a gene's codedinformation is converted into structures present and operating in acell.

“Nucleobase sequence” means the order of contiguous nucleobases in anoligomeric compound or nucleic acid, typically listed in a 5′ to 3′orientation, independent of any sugar, linkage, and/or nucleobasemodification.

“Contiguous nucleobases” means nucleobases immediately adjacent to eachother in a nucleic acid.

“Nucleobase complementarity” means the ability of two nucleobases topair non-covalently via hydrogen bonding.

“Complementary” means that one nucleic acid is capable of hybridizing toanother nucleic acid or oligonucleotide. In certain embodiments,complementary refers to an oligonucleotide capable of hybridizing to atarget nucleic acid.

“Fully complementary” means each nucleobase of an oligonucleotide iscapable of pairing with a nucleobase at each corresponding position in atarget nucleic acid. In certain embodiments, an oligonucleotide is fullycomplementary to a microRNA, i.e. each nucleobase of the oligonucleotideis complementary to a nucleobase at a corresponding position in themicroRNA. In certain embodiments, an oligonucleotide wherein eachnucleobase has complementarity to a nucleobase within a region of amicroRNA stem-loop sequence is fully complementary to the microRNAstem-loop sequence.

“Percent complementarity” means the percentage of nucleobases of anoligonucleotide that are complementary to an equal-length portion of atarget nucleic acid. Percent complementarity is calculated by dividingthe number of nucleobases of the oligonucleotide that are complementaryto nucleobases at corresponding positions in the target nucleic acid bythe total number of nucleobases in the oligonucleotide.

“Percent identity” means the number of nucleobases in a first nucleicacid that are identical to nucleobases at corresponding positions in asecond nucleic acid, divided by the total number of nucleobases in thefirst nucleic acid. In certain embodiments, the first nucleic acid is amicroRNA and the second nucleic acid is a microRNA. In certainembodiments, the first nucleic acid is an oligonucleotide and the secondnucleic acid is an oligonucleotide.

“Hybridize” means the annealing of complementary nucleic acids thatoccurs through nucleobase complementarity.

“Mismatch” means a nucleobase of a first nucleic acid that is notcapable of Watson-Crick pairing with a nucleobase at a correspondingposition of a second nucleic acid.

“Identical” in the context of nucleobase sequences, means having thesame nucleobase sequence, independent of sugar, linkage, and/ornucleobase modifications and independent of the methyl state of anypyrimidines present.

“MicroRNA” means an endogenous non-coding RNA between 18 and 25nucleobases in length, which is the product of cleavage of apre-microRNA by the enzyme Dicer. Examples of mature microRNAs are foundin the microRNA database known as miRBase(http://microrna.sanger.ac.uk/). In certain embodiments, microRNA isabbreviated as “microRNA” or “miR.”

“Pre-microRNA” or “pre-miR” means a non-coding RNA having a hairpinstructure, which is the product of cleavage of a pri-miR by thedouble-stranded RNA-specific ribonuclease known as Drosha.

“Stem-loop sequence” means an RNA having a hairpin structure andcontaining a mature microRNA sequence. Pre-microRNA sequences andstem-loop sequences may overlap. Examples of stem-loop sequences arefound in the microRNA database known as miRBase(http://microrna.sanger.ac.uk/).

“Pri-microRNA” or “pri-miR” means a non-coding RNA having a hairpinstructure that is a substrate for the double-stranded RNA-specificribonuclease Drosha.

“microRNA precursor” means a transcript that originates from a genomicDNA and that comprises a non-coding, structured RNA comprising one ormore microRNA sequences. For example, in certain embodiments a microRNAprecursor is a pre-microRNA. In certain embodiments, a microRNAprecursor is a pri-microRNA.

“microRNA-regulated transcript” means a transcript that is regulated bya microRNA.

“Seed sequence” means a nucleobase sequence comprising from 6 to 8contiguous nucleobases of nucleobases 1 to 9 of the 5′-end of a maturemicroRNA sequence.

“Seed match sequence” means a nucleobase sequence that is complementaryto a seed sequence, and is the same length as the seed sequence.

“Oligomeric compound” means a compound that comprises a plurality oflinked monomeric subunits. Oligomeric compounds includedoligonucleotides.

“Oligonucleotide” means a compound comprising a plurality of linkednucleosides, each of which can be modified or unmodified, independentfrom one another.

“Naturally occurring internucleoside linkage” means a 3′ to 5′phosphodiester linkage between nucleosides.

“Natural sugar” means a sugar found in DNA (2′-H) or RNA (2′-OH).

“Internucleoside linkage” means a covalent linkage between adjacentnucleosides.

“Linked nucleosides” means nucleosides joined by a covalent linkage.

“Nucleobase” means a heterocyclic moiety capable of non-covalentlypairing with another nucleobase.

“Nucleoside” means a nucleobase linked to a sugar moiety.

“Nucleotide” means a nucleoside having a phosphate group covalentlylinked to the sugar portion of a nucleoside.

“Compound comprising a modified oligonucleotide consisting of” a numberof linked nucleosides means a compound that includes a modifiedoligonucleotide having the specified number of linked nucleosides. Thus,the compound may include additional substituents or conjugates. Unlessotherwise indicated, the compound does not include any additionalnucleosides beyond those of the modified oligonucleotide.

“Modified oligonucleotide” means an oligonucleotide having one or moremodifications relative to a naturally occurring terminus, sugar,nucleobase, and/or internucleoside linkage. A modified oligonucleotidemay comprise unmodified nucleosides.

“Single-stranded modified oligonucleotide” means a modifiedoligonucleotide which is not hybridized to a complementary strand.

“Modified nucleoside” means a nucleoside having any change from anaturally occurring nucleoside. A modified nucleoside may have amodified sugar and an unmodified nucleobase. A modified nucleoside mayhave a modified sugar and a modified nucleobase. A modified nucleosidemay have a natural sugar and a modified nucleobase. In certainembodiments, a modified nucleoside is a bicyclic nucleoside. In certainembodiments, a modified nucleoside is a non-bicyclic nucleoside.

“Modified internucleoside linkage” means any change from a naturallyoccurring internucleoside linkage.

“Phosphorothioate internucleoside linkage” means a linkage betweennucleosides where one of the non-bridging atoms is a sulfur atom.

“Modified sugar moiety” means substitution and/or any change from anatural sugar.

“Unmodified nucleobase” means the naturally occurring heterocyclic basesof RNA or DNA: the purine bases adenine (A) and guanine (G), and thepyrimidine bases thymine (T), cytosine (C) (including 5-methylcytosine),and uracil (U).

“5-methylcytosine” means a cytosine comprising a methyl group attachedto the 5 position.

“Non-methylated cytosine” means a cytosine that does not have a methylgroup attached to the 5 position.

“Modified nucleobase” means any nucleobase that is not an unmodifiednucleobase.

“Sugar moiety” means a naturally occurring furanosyl or a modified sugarmoiety.

“Modified sugar moiety” means a substituted sugar moiety or a sugarsurrogate.

“2′-O-methyl sugar” or “2′-OMe sugar” means a sugar having a O-methylmodification at the 2′ position.

“2′-O-methoxyethyl sugar” or “2′-MOE sugar” means a sugar having aO-methoxyethyl modification at the 2′ position.

“2′-fluoro” or “2′-F” means a sugar having a fluoro modification of the2′ position.

“Bicyclic sugar moiety” means a modified sugar moiety comprising a 4 to7 membered ring (including by not limited to a furanosyl) comprising abridge connecting two atoms of the 4 to 7 membered ring to form a secondring, resulting in a bicyclic structure. In certain embodiments, the 4to 7 membered ring is a sugar ring. In certain embodiments the 4 to 7membered ring is a furanosyl. In certain such embodiments, the bridgeconnects the 2′-carbon and the 4′-carbon of the furanosyl. Nonlimitingexemplary bicyclic sugar moieties include LNA, ENA, cEt, S-cEt, andR-cEt.

“Locked nucleic acid (LNA) sugar moiety” means a substituted sugarmoiety comprising a (CH₂)—O bridge between the 4′ and 2′ furanose ringatoms.

“ENA sugar moiety” means a substituted sugar moiety comprising a(CH₂)₂—O bridge between the 4′ and 2′ furanose ring atoms.

“Constrained ethyl (cEt) sugar moiety” means a substituted sugar moietycomprising a CH(CH₃)—O bridge between the 4′ and the 2′ furanose ringatoms. In certain embodiments, the CH(CH₃)—O bridge is constrained inthe S orientation. In certain embodiments, the (CH₂)₂—O is constrainedin the R orientation.

“S-cEt sugar moiety” means a substituted sugar moiety comprising anS-constrained CH(CH₃)—O bridge between the 4′ and the 2′ furanose ringatoms.

“R-cEt sugar moiety” means a substituted sugar moiety comprising anR-constrained CH(CH₃)—O bridge between the 4′ and the 2′ furanose ringatoms.

“2′-O-methyl nucleoside” means a 2′-modified nucleoside having a2′-O-methyl sugar modification.

“2′-O-methoxyethyl nucleoside” means a 2′-modified nucleoside having a2′-O-methoxyethyl sugar modification. A 2′-O-methoxyethyl nucleoside maycomprise a modified or unmodified nucleobase.

“2′-fluoro nucleoside” means a 2′-modified nucleoside having a 2′-fluorosugar modification. A 2′-fluoro nucleoside may comprise a modified orunmodified nucleobase.

“Bicyclic nucleoside” means a 2′-modified nucleoside having a bicyclicsugar moiety. A bicyclic nucleoside may have a modified or unmodifiednucleobase.

“cEt nucleoside” means a nucleoside comprising a cEt sugar moiety. A cEtnucleoside may comprise a modified or unmodified nucleobase.

“S-cEt nucleoside” means a nucleoside comprising an S-cEt sugar moiety.

“R-cEt nucleoside” means a nucleoside comprising an R-cEt sugar moiety.

“β-D-deoxyribonucleoside” means a naturally occurring DNA nucleoside.

“β-D-ribonucleoside” means a naturally occurring RNA nucleoside.

“LNA nucleoside” means a nucleoside comprising a LNA sugar moiety.

“ENA nucleoside” means a nucleoside comprising an ENA sugar moiety.

“Subject” means a human or non-human animal selected for treatment ortherapy.

“Subject in need thereof” means a subject that is identified as in needof a therapy or treatment.

“Subject suspected of having” means a subject exhibiting one or moreclinical indicators of a disease.

“Administering” means providing a pharmaceutical agent or composition toa subject, and includes, but is not limited to, administering by amedical professional and self-administering.

“Parenteral administration” means administration through injection orinfusion. Parenteral administration includes, but is not limited to,subcutaneous administration, intravenous administration, andintramuscular administration.

“Subcutaneous administration” means administration just below the skin.

“Intravenous administration” means administration into a vein.

“Administered concomitantly” refers to the co-administration of two ormore agents in any manner in which the pharmacological effects of bothare manifest in the patient at the same time. Concomitant administrationdoes not require that both agents be administered in a singlepharmaceutical composition, in the same dosage form, or by the sameroute of administration. The effects of both agents need not manifestthemselves at the same time. The effects need only be overlapping for aperiod of time and need not be coextensive.

“Duration” means the period of time during which an activity or eventcontinues. In certain embodiments, the duration of treatment is theperiod of time during which doses of a pharmaceutical agent orpharmaceutical composition are administered.

“Therapy” means a disease treatment method. In certain embodiments,therapy includes, but is not limited to, chemotherapy, radiationtherapy, or administration of a pharmaceutical agent.

“Treatment” means the application of one or more specific proceduresused for the cure or amelioration of a disease. In certain embodiments,the specific procedure is the administration of one or morepharmaceutical agents.

“Ameliorate” means to lessen the severity of at least one indicator of acondition or disease. In certain embodiments, amelioration includes adelay or slowing in the progression of one or more indicators of acondition or disease. The severity of indicators may be determined bysubjective or objective measures which are known to those skilled in theart.

“At risk for developing” means the state in which a subject ispredisposed to developing a condition or disease. In certainembodiments, a subject at risk for developing a condition or diseaseexhibits one or more symptoms of the condition or disease, but does notexhibit a sufficient number of symptoms to be diagnosed with thecondition or disease. In certain embodiments, a subject at risk fordeveloping a condition or disease exhibits one or more symptoms of thecondition or disease, but to a lesser extent required to be diagnosedwith the condition or disease.

“Prevent the onset of” means to prevent the development of a conditionor disease in a subject who is at risk for developing the disease orcondition. In certain embodiments, a subject at risk for developing thedisease or condition receives treatment similar to the treatmentreceived by a subject who already has the disease or condition.

“Delay the onset of” means to delay the development of a condition ordisease in a subject who is at risk for developing the disease orcondition. In certain embodiments, a subject at risk for developing thedisease or condition receives treatment similar to the treatmentreceived by a subject who already has the disease or condition.

“Therapeutic agent” means a pharmaceutical agent used for the cure,amelioration or prevention of a disease.

“Dose” means a specified quantity of a pharmaceutical agent provided ina single administration. In certain embodiments, a dose may beadministered in two or more boluses, tablets, or injections. Forexample, in certain embodiments, where subcutaneous administration isdesired, the desired dose requires a volume not easily accommodated by asingle injection. In such embodiments, two or more injections may beused to achieve the desired dose. In certain embodiments, a dose may beadministered in two or more injections to minimize injection sitereaction in an individual. In certain embodiments, a dose isadministered as a slow infusion.

“Dosage unit” means a form in which a pharmaceutical agent is provided.In certain embodiments, a dosage unit is a vial containing lyophilizedoligonucleotide. In certain embodiments, a dosage unit is a vialcontaining reconstituted oligonucleotide.

“Therapeutically effective amount” refers to an amount of apharmaceutical agent that provides a therapeutic benefit to an animal.

“Pharmaceutical composition” means a mixture of substances suitable foradministering to an individual that includes a pharmaceutical agent. Forexample, a pharmaceutical composition may comprise a sterile aqueoussolution.

“Pharmaceutical agent” means a substance that provides a therapeuticeffect when administered to a subject.

“Active pharmaceutical ingredient” means the substance in apharmaceutical composition that provides a desired effect.

“Improved organ function” means a change in organ function toward normallimits. In certain embodiments, organ function is assessed by measuringmolecules found in a subject's blood or urine. For example, in certainembodiments, improved kidney function is measured by a reduction inblood urea nitrogen, a reduction in proteinuria, a reduction inalbuminuria, etc.

“Acceptable safety profile” means a pattern of side effects that iswithin clinically acceptable limits.

“Side effect” means a physiological response attributable to a treatmentother than desired effects. In certain embodiments, side effectsinclude, without limitation, injection site reactions, liver functiontest abnormalities, kidney function abnormalities, liver toxicity, renaltoxicity, central nervous system abnormalities, and myopathies. Suchside effects may be detected directly or indirectly. For example,increased aminotransferase levels in serum may indicate liver toxicityor liver function abnormality. For example, increased bilirubin mayindicate liver toxicity or liver function abnormality.

“Subject compliance” means adherence to a recommended or prescribedtherapy by a subject.

“Comply” means the adherence with a recommended therapy by a subject.

“Recommended therapy” means a treatment recommended by a medicalprofessional for the treatment, amelioration, or prevention of adisease.

The term “blood” as used herein, encompasses whole blood and bloodfractions, such as serum and plasma.

Overview

Alport Syndrome is an inherited form of kidney disease in which anabnormal level of glomerular basement membrane (GBM) is produced,leading to interstitial fibrosis, glomerular sclerosis and typicallyleads to end-stage renal disease. In the management of Alport Syndrome,the primary goal for treatment is to maintain kidney function andprevent the onset of end-stage renal disease (ESRD), which in turnimproves life expectancy of subjects with Alport Syndrome.

Alport Syndrome is characterized by progressive fibrosis due to defectsin GBM composition, thus improvements in GBM morphology and function aredesirable. It is demonstrated herein that modified oligonucleotidetargeted to miR-21 improves kidney function in an experimental model ofAlport Syndrome. Additionally, glomerular sclerosis and fibrosis arereduced following anti-miR-21 treatment. It is further demonstratedherein that anti-miR-21 improves survival in an experimental model ofAlport Syndrome. As such, these modified oligonucleotides targeted tomiR-21 are useful for the treatment of Alport Syndrome.

Certain Uses of the Invention

Provided herein are methods for the treatment of Alport Syndrome,comprising administering to a subject having or suspected of havingAlport Syndrome a modified oligonucleotide complementary to miR-21.

In certain embodiments, the subject has been diagnosed as having AlportSyndrome prior to administration of the modified oligonucleotide.Diagnosis of Alport Syndrome may be achieved through evaluation ofparameters including, without limitation, a subject's family history,clinical features (including without limitation proteinuria,albuminuria, hematuria, impaired GFR, deafness and/or ocular changes)and results of tissue biopsies. Kidney biopsies may be tested for thepresence or absence of the type IV collagen alpha-3, alpha-4, andalpha-5 chains. Additionally, structural changes in the glomerulus canbe detected by electron microscopy of kidney biopsy material. A skinbiopsy may be tested for the presence of the type IV collagen alpha-5chain, which is normally present in skin and almost always absent frommale subjects with the X-linked form of Alport Syndrome. Diagnosis ofAlport Syndrome may also include screening for mutations in one or moreof the Col4a3, Col4a4, or Col4a5 genes.

In certain embodiments, levels of miR-21 are increased in the kidney ofa subject having Alport Syndrome. In certain embodiments, prior toadministration, a subject is determined to have an increased level ofmiR-21 in the kidney. miR-21 levels may be measured from kidney biopsymaterial. In certain embodiments, prior to administration, a subject isdetermined to have an increased level of miR-21 in the urine or blood ofthe subject.

In certain embodiments, administration of a modified oligonucleotidecomplementary to miR-21 results in one or more clinically beneficialoutcomes. In certain embodiments, the administration improves kidneyfunction. In certain embodiments, the administration delays the onset ofend-stage renal disease. In certain embodiments, the administrationdelays time to dialysis. In certain embodiments, the administrationdelays time to renal transplant. In certain embodiments, theadministration improves life expectancy of the subject.

In certain embodiments, the administering reduces kidney fibrosis. Incertain embodiments the administering slows further progression ofkidney fibrosis. In certain embodiments, the administration haltsfurther progression of kidney fibrosis. In certain embodiments, theadministration reduces hematuria. In certain embodiments, theadministration delays the onset of hematuria. In certain embodiments,the administration reduces proteinuria. In certain embodiments, theadministration delays the onset of proteinuria.

The subject having or suspected of having Alport Syndrome may have amutation in the gene encoding the alpha 3 chain of type IV collagen(Col4a3), a mutation in the gene encoding the alpha 4 chain of type IVcollagen (Col4a4), or a mutation in the gene encoding the alpha 5 chainof type IV collagen (Col4a5). In certain embodiments, the subject ismale. In certain embodiments, the subject is female.

In certain embodiments the subject has impaired kidney function. Incertain embodiments, the subject is in need of improved kidney function.In certain embodiments, the subject is identified as having impairedkidney function. In certain embodiments, the subject is identified ashaving hematuria. In certain embodiments, the subject is identified ashaving proteinuria.

In any of the embodiments provided herein, a subject may be subjected tocertain tests to evaluate kidney function. Such tests include, withoutlimitation, measurement of blood urea nitrogen in the subject; measuringcreatinine in the blood of the subject; measuring creatinine clearancein the blood of the subject; measuring proteinuria in the subject;measuring albumin:creatinine ratio in the subject; measuring glomerularfiltration rate in the subject; and measuring urinary output in thesubject.

In any of the embodiments provided herein, proteins present in the urineor blood may be used to evaluate kidney function. Such tests of kidneyfunction include, but are not limited to, measuringN-acetyl-β-D-glucosaminidase (NAG) protein in the urine of the subject;measuring neutrophil gelatinase-associated lipocalin (NGAL) protein inthe urine of the subject; measuring kidney injury molecule-1 (KIM-1)protein in the urine of the subject; measuring interleukin-18 (IL-18)protein in the urine of the subject; measuring connective tissue growthfactor (CTGF) levels in the urine of the subject; measuring monocytechemoattractant protein 1 (MCP1) levels in the urine of the subject;measuring collagen IV (Col IV) fragments in the urine of the subject;measuring collagen III (Col III) fragment levels in the urine of thesubject; measuring cystatin C protein in the blood of a subject;measuring β-trace protein (BTP) in the blood of a subject; and measuring2-microglobulin (B2M) in the blood of a subject. In any of theembodiments provided herein, markers of podocyte injury can be measuringin the urine. Such proteins include nephrin and podocin. The proteinsmay be quantitated, for example, by enzyme-linked immunosorbent assay(ELISA), or radioimmunoassay (RIA) using commercially available kits.

In any of the embodiments provided herein, the administration of amodified oligonucleotide targeted to miR-21 improves one or more markersof kidney function in the subject. Improvements in markers of kidneyfunction include, without limitation: reduced blood urea nitrogen in thesubject; reduced creatinine in the blood of the subject; improvedcreatinine clearance in the subject; reduced proteinuria in the subject;reduced albumin:creatinine ratio in the subject; improved glomerularfiltration rate in the subject; and/or increased urinary output in thesubject.

Certain Additional Therapies

Treatments for Alport Syndrome or any of the conditions listed hereinmay comprise more than one therapy. As such, in certain embodimentsprovided herein are methods for treating a subject having or suspectedof having Alport Syndrome comprising administering at least one therapyin addition to administering a modified oligonucleotide having anucleobase sequence complementary to a miR-21.

In certain embodiments, the at least one additional therapy comprises apharmaceutical agent.

In certain embodiments, pharmaceutical agents include angiotensin IIreceptor blockers (ARB). In certain embodiments, an angiotensin IIreceptor blocker is candesartan, irbesartan, olmesartan, losartan,valsartan, telmisartan, or eprosartan.

In certain embodiments, pharmaceutical agents include angiotensin IIconverting enzyme (ACE) inhibitors. In certain embodiments, an ACEinhibitor is captopril, enalapril, lisinopril, benazepril, quinapril,fosinopril, or ramipril.

In certain embodiments, a pharmaceutical agent is an anti-hypertensiveagent. Anti-hypertensive agents are used to control blood pressure ofthe subject.

In certain embodiments, a pharmaceutical agent is a vitamin D analog.Vitamin D analogs may be used to limit the production of parathyroidhormone in the subject.

In certain embodiments, a pharmaceutical agent is an oral phosphatebinder that reduces dietary phosphate absorption.

In certain embodiments, pharmaceutical agents include immunosuppressiveagents. In certain embodiments, an immunosuppressive agent is acorticosteroid, cyclophosphamide, or mycophenolate mofetil.

In certain embodiments, a pharmaceutical agent is cyclosporine, anHMG-Coenzyme A inhibitor, a vasopeptidase inhibitor, or aTGF-beta-antagonist.

In certain embodiments, an additional therapy is gene therapy. Incertain embodiments, the gene therapy provides a normal Col4a3 gene. Incertain embodiments, the gene therapy provides a normal Col4a4 gene. Incertain embodiments, the gene therapy provides a normal Col4a5 gene.

In certain embodiments, an additional therapy is dialysis. In certainembodiments, an additional therapy is renal transplant.

In certain embodiments, pharmaceutical agents include anti-inflammatoryagents. In certain embodiments, an anti-inflammatory agent is asteroidal anti-inflammatory agent. In certain embodiments, a steroidanti-inflammatory agent is a corticosteroid. In certain embodiments, acorticosteroid is prednisone. In certain embodiments, ananti-inflammatory agent is a non-steroidal anti-inflammatory drug. Incertain embodiments, a non-steroidal anti-inflammatory agent isibuprofen, a COX-I inhibitor, or a COX-2 inhibitor.

In certain embodiments, a pharmaceutical agent is a pharmaceutical agentthat blocks one or more responses to fibrogenic signals.

In certain embodiments, pharmaceutical agents include anti-diabeticagent. Antidiabetic agents include, but are not limited to, biguanides,glucosidase inhibitors, insulins, sulfonylureas, and thiazolidenediones.

Certain MicroRNA Nucleobase Sequences

The modified oligonucleotides described herein have a nucleobasesequence that is complementary to miR-21 (SEQ ID NO: 1), or a precursorthereof (SEQ ID NO: 2). In certain embodiments, each nucleobase of themodified oligonucleotide is capable of undergoing base-pairing with anucleobase at each corresponding position in the nucleobase sequence ofmiR-21, or a precursor thereof. In certain embodiments the nucleobasesequence of a modified oligonucleotide may have one or more mismatchedbase pairs with respect to the nucleobase sequence of miR-21 orprecursor sequence, and remains capable of hybridizing to its targetsequence.

As the miR-21 sequence is contained within the miR-21 precursorsequence, a modified oligonucleotide having a nucleobase sequencecomplementary to miR-21 is also complementary to a region of the miR-21precursor.

In certain embodiments, a modified oligonucleotide consists of a numberof linked nucleosides that is equal to the length of miR-21.

In certain embodiments, the number of linked nucleosides of a modifiedoligonucleotide is less than the length of miR-21. A modifiedoligonucleotide having a number of linked nucleosides that is less thanthe length of miR-21, wherein each nucleobase of the modifiedoligonucleotide is complementary to each nucleobase at a correspondingposition of miR-21, is considered to be a modified oligonucleotidehaving a nucleobase sequence that is fully complementary to a region ofthe miR-21 sequence. For example, a modified oligonucleotide consistingof 19 linked nucleosides, where each nucleobase is complementary to acorresponding position of miR-21 that is 22 nucleobases in length, isfully complementary to a 19 nucleobase region of miR-21. Such a modifiedoligonucleotide has 100% complementarity to a 19 nucleobase portion ofmiR-21, and is considered to be 100% complementary to miR-21.

In certain embodiments, a modified oligonucleotide comprises anucleobase sequence that is complementary to a seed sequence, i.e. amodified oligonucleotide comprises a seed-match sequence. In certainembodiments, a seed sequence is a hexamer seed sequence. In certain suchembodiments, a seed sequence is nucleobases 1-6 of miR-21. In certainsuch embodiments, a seed sequence is nucleobases 2-7 of miR-21. Incertain such embodiments, a seed sequence is nucleobases 3-8 of miR-21.In certain embodiments, a seed sequence is a heptamer seed sequence. Incertain such embodiments, a heptamer seed sequence is nucleobases 1-7 ofmiR-21. In certain such embodiments, a heptamer seed sequence isnucleobases 2-8 of miR-21. In certain embodiments, the seed sequence isan octamer seed sequence. In certain such embodiments, an octamer seedsequence is nucleobases 1-8 of miR-21. In certain embodiments, anoctamer seed sequence is nucleobases 2-9 of miR-21.

In certain embodiments, a modified oligonucleotide has a nucleobasesequence having one mismatch with respect to the nucleobase sequence ofmiR-21, or a precursor thereof. In certain embodiments, a modifiedoligonucleotide has a nucleobase sequence having two mismatches withrespect to the nucleobase sequence of miR-21, or a precursor thereof. Incertain such embodiments, a modified oligonucleotide has a nucleobasesequence having no more than two mismatches with respect to thenucleobase sequence of miR-21, or a precursor thereof. In certain suchembodiments, the mismatched nucleobases are contiguous. In certain suchembodiments, the mismatched nucleobases are not contiguous.

In certain embodiments, the number of linked nucleosides of a modifiedoligonucleotide is greater than the length of miR-21. In certain suchembodiments, the nucleobase of an additional nucleoside is complementaryto a nucleobase of the miR-21 stem-loop sequence. In certainembodiments, the number of linked nucleosides of a modifiedoligonucleotide is one greater than the length of miR-21. In certainsuch embodiments, the additional nucleoside is at the 5′ terminus of anoligonucleotide. In certain such embodiments, the additional nucleosideis at the 3′ terminus of an oligonucleotide. In certain embodiments, thenumber of linked nucleosides of a modified oligonucleotide is twogreater than the length of miR-21. In certain such embodiments, the twoadditional nucleosides are at the 5′ terminus of an oligonucleotide. Incertain such embodiments, the two additional nucleosides are at the 3′terminus of an oligonucleotide. In certain such embodiments, oneadditional nucleoside is located at the 5′ terminus and one additionalnucleoside is located at the 3′ terminus of an oligonucleotide. Incertain embodiments, a region of the oligonucleotide may be fullycomplementary to the nucleobase sequence of miR-21, but the entiremodified oligonucleotide is not fully complementary to miR-21. Forexample, a modified oligonucleotide consisting of 24 linked nucleosides,where the nucleobases of nucleosides 1 through 22 are each complementaryto a corresponding position of miR-21 that is 22 nucleobases in length,has a 22 nucleoside portion that is fully complementary to thenucleobase sequence of miR-21 and approximately 92% overallcomplementarity to the nucleobase sequence of miR-21.

Certain Modified Oligonucleotides

In certain embodiments, a modified oligonucleotide has the structure5′-A_(E)C_(S)ATC_(S)AGTC_(S)TGAU_(S)AAGC_(S)TA_(E)-3′ (SEQ ID NO: 3),where nucleosides not followed by a subscript indicateβ-D-deoxyribonucleosides; nucleosides followed by a subscript “E”indicate 2′-MOE nucleosides; nucleosides followed by a subscript “S”indicate S-cEt nucleosides; and each internucleoside linkage is aphosphorothioate internucleoside linkage.

In certain embodiments, a modified oligonucleotide has the structure5′-A_(E)C_(S)ATC_(S)A_(S)GTC_(S)U_(S)GAU_(S)A_(S)AGC_(S)UsA_(E)-3′ (SEQID NO: 3), where nucleosides not followed by a subscript indicateβ-D-deoxyribonucleosides; nucleosides followed by a subscript “E”indicate 2′-MOE nucleosides; nucleosides followed by a subscript “S”indicate S-cEt nucleosides; and each internucleoside linkage is aphosphorothioate internucleoside linkage.

In certain embodiments, a modified oligonucleotide has the structure5′-^(Me)C_(E)A_(S)A_(S)T_(E)C_(S)U_(S)A_(E)A_(E)U_(S)A_(S)A_(E)G_(E)C_(S)T_(E)A_(S)-3′(SEQ ID NO: 4), where nucleosides not followed by a subscript indicateβ-D-deoxyribonucleosides; nucleosides followed by a subscript “E”indicate 2′-MOE nucleosides; nucleosides followed by a subscript “S”indicate S-cEt nucleosides; a superscript “Me” indicates a 5-methylgroup on the base of the nucleoside; and each internucleoside linkage isa phosphorothioate internucleoside linkage.

In certain embodiments, a modified oligonucleotide has the structure5′-A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TGAU_(S)AAGC_(S)U_(S)A_(S)-3′(SEQ ID NO: 3), where nucleosides not followed by a subscript indicateβ-D-deoxyribonucleosides; nucleosides followed by a subscript “E”indicate 2′-MOE nucleosides; nucleosides followed by a subscript “S”indicate S-cEt nucleosides; and each internucleoside linkage is aphosphorothioate internucleoside linkage.

In certain embodiments, a modified oligonucleotide comprises one or more5-methylcytosines. In certain embodiments, each cytosine of a modifiedoligonucleotide comprises a 5-methylcytosine.

In certain embodiments, a modified oligonucleotide consists of 8 to 30linked nucleosides. In certain embodiments, a modified oligonucleotideconsists of 12 to 25 linked nucleosides. In certain embodiments, amodified oligonucleotide consists of 15 to 30 linked nucleosides. Incertain embodiments, a modified oligonucleotide consists of 15 to 25linked nucleosides. In certain embodiments, a modified oligonucleotideconsists of 15 to 19 linked nucleosides. In certain embodiments, amodified oligonucleotide consists of 15 to 16 linked nucleosides. Incertain embodiments, a modified oligonucleotide consists of 19 to 24linked nucleosides. In certain embodiments, a modified oligonucleotideconsists of 21 to 24 linked nucleosides.

In certain embodiments, a modified oligonucleotide consists of 8 linkednucleosides. In certain embodiments, a modified oligonucleotide consistsof 9 linked nucleosides. In certain embodiments, a modifiedoligonucleotide consists of 10 linked nucleosides. In certainembodiments, a modified oligonucleotide consists of 11 linkednucleosides. In certain embodiments, a modified oligonucleotide consistsof 12 linked nucleosides. In certain embodiments, a modifiedoligonucleotide consists of 13 linked nucleosides. In certainembodiments, a modified oligonucleotide consists of 14 linkednucleosides. In certain embodiments, a modified oligonucleotide consistsof 15 linked nucleosides. In certain embodiments, a modifiedoligonucleotide consists of 16 linked nucleosides. In certainembodiments, a modified oligonucleotide consists of 17 linkednucleosides. In certain embodiments, a modified oligonucleotide consistsof 18 linked nucleosides. In certain embodiments, a modifiedoligonucleotide consists of 19 linked nucleosides. In certainembodiments, a modified oligonucleotide consists of 20 linkednucleosides. In certain embodiments, a modified oligonucleotide consistsof 21 linked nucleosides. In certain embodiments, a modifiedoligonucleotide consists of 22 linked nucleosides. In certainembodiments, a modified oligonucleotide consists of 23 linkednucleosides. In certain embodiments, a modified oligonucleotide consistsof 24 linked nucleosides. In certain embodiments, a modifiedoligonucleotide consists of 25 linked nucleosides.

Certain Modifications

In certain embodiments, oligonucleotides provided herein may compriseone or more modifications to a nucleobase, sugar, and/or internucleosidelinkage, and as such is a modified oligonucleotide. A modifiednucleobase, sugar, and/or internucleoside linkage may be selected overan unmodified form because of desirable properties such as, for example,enhanced cellular uptake, enhanced affinity for other oligonucleotidesor nucleic acid targets and increased stability in the presence ofnucleases.

In certain embodiments, a modified oligonucleotide comprises one or moremodified nucleosides. In certain such embodiments, a modified nucleosideis a stabilizing nucleoside. An example of a stabilizing nucleoside is asugar-modified nucleoside.

In certain embodiments, a modified nucleoside is a sugar-modifiednucleoside. In certain such embodiments, the sugar-modified nucleosidescan further comprise a natural or modified heterocyclic base moietyand/or a natural or modified internucleoside linkage and may includefurther modifications independent from the sugar modification. Incertain embodiments, a sugar modified nucleoside is a 2′-modifiednucleoside, wherein the sugar ring is modified at the 2′ carbon fromnatural ribose or 2′-deoxy-ribose.

In certain embodiments, a 2′-modified nucleoside has a bicyclic sugarmoiety. In certain such embodiments, the bicyclic sugar moiety is a Dsugar in the alpha configuration. In certain such embodiments, thebicyclic sugar moiety is a D sugar in the beta configuration. In certainsuch embodiments, the bicyclic sugar moiety is an L sugar in the alphaconfiguration. In certain such embodiments, the bicyclic sugar moiety isan L sugar in the beta configuration.

Nucleosides comprising such bicyclic sugar moieties are referred to asbicyclic nucleosides or BNAs. In certain embodiments, bicyclicnucleosides include, but are not limited to, (A) α-L-Methyleneoxy(4′-CH₂—O-2′) BNA; (B) β-D-Methyleneoxy (4′-CH₂—O-2′) BNA; (C)Ethyleneoxy (4′-(CH₂)₂—O-2′) BNA; (D) Aminooxy (4′-CH₂—O—N(R)—2′) BNA;(E) Oxyamino (4′-CH₂—N(R)—O-2′) BNA; (F) Methyl(methyleneoxy)(4′-CH(CH₃)—O-2′) BNA (also referred to as constrained ethyl or cEt);(G) methylene-thio (4′-CH₂—S-2′) BNA; (H) methylene-amino(4′-CH2-N(R)—2′) BNA; (I) methyl carbocyclic (4′-CH₂—CH(CH₃)—2′) BNA;(J) c-MOE (4′-CH₂—OMe-2′) BNA and (K) propylene carbocyclic(4′-(CH₂)₃-2′) BNA as depicted below.

wherein Bx is a nucleobase moiety and R is, independently, H, aprotecting group, or C₁-C₁₂ alkyl.

In certain embodiments, a 2′-modified nucleoside comprises a2′-substituent group selected from F, OCF₃, O—CH₃, OCH₂CH₂OCH₃,2′-O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(CH₃)₂, —O(CH₂)₂O(CH₂)₂N—(CH₃)₂, andO—CH₂—C(═O)—N(H)CH₃.

In certain embodiments, a 2′-modified nucleoside comprises a2′-substituent group selected from F, O—CH₃, and OCH₂CH₂OCH₃.

In certain embodiments, a sugar-modified nucleoside is a 4′-thiomodified nucleoside. In certain embodiments, a sugar-modified nucleosideis a 4′-thio-2′-modified nucleoside. A 4′-thio modified nucleoside has aβ-D-ribonucleoside where the 4′-0 replaced with 4′-S. A4′-thio-2′-modified nucleoside is a 4′-thio modified nucleoside havingthe 2′-OH replaced with a 2′-substituent group. Suitable 2′-substituentgroups include 2′-OCH₃, 2′-O—(CH₂)₂—OCH₃, and 2′-F.

In certain embodiments, a modified oligonucleotide comprises one or moreinternucleoside modifications. In certain such embodiments, eachinternucleoside linkage of a modified oligonucleotide is a modifiedinternucleoside linkage. In certain embodiments, a modifiedinternucleoside linkage comprises a phosphorus atom.

In certain embodiments, a modified oligonucleotide comprises at leastone phosphorothioate internucleoside linkage. In certain embodiments,each internucleoside linkage of a modified oligonucleotide is aphosphorothioate internucleoside linkage.

In certain embodiments, a modified oligonucleotide comprises one or moremodified nucleobases. In certain embodiments, a modified nucleobase isselected from 5-hydroxymethyl cytosine, 7-deazaguanine and7-deazaadenine. In certain embodiments, a modified nucleobase isselected from 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and2-pyridone. In certain embodiments, a modified nucleobase is selectedfrom 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2 aminopropyladenine, 5-propynyluraciland 5-propynylcytosine.

In certain embodiments, a modified nucleobase comprises a polycyclicheterocycle. In certain embodiments, a modified nucleobase comprises atricyclic heterocycle. In certain embodiments, a modified nucleobasecomprises a phenoxazine derivative. In certain embodiments, thephenoxazine can be further modified to form a nucleobase known in theart as a G-clamp.

In certain embodiments, a modified oligonucleotide is conjugated to oneor more moieties which enhance the activity, cellular distribution orcellular uptake of the resulting antisense oligonucleotides. In certainsuch embodiments, the moiety is a cholesterol moiety. In certainembodiments, the moiety is a lipid moiety. Additional moieties forconjugation include carbohydrates, phospholipids, biotin, phenazine,folate, phenanthridine, anthraquinone, acridine, fluoresceins,rhodamines, coumarins, and dyes. In certain embodiments, thecarbohydrate moiety is N-acetyl-D-galactosamine (GalNac). In certainembodiments, a conjugate group is attached directly to anoligonucleotide. In certain embodiments, a conjugate group is attachedto a modified oligonucleotide by a linking moiety selected from amino,hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double or triplebonds), 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), 6-aminohexanoicacid (AHEX or AHA), substituted C1-C10 alkyl, substituted orunsubstituted C2-C10 alkenyl, and substituted or unsubstituted C2-C10alkynyl. In certain such embodiments, a substituent group is selectedfrom hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol,thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

In certain such embodiments, the compound comprises a modifiedoligonucleotide having one or more stabilizing groups that are attachedto one or both termini of a modified oligonucleotide to enhanceproperties such as, for example, nuclease stability. Included instabilizing groups are cap structures. These terminal modificationsprotect a modified oligonucleotide from exonuclease degradation, and canhelp in delivery and/or localization within a cell. The cap can bepresent at the 5′-terminus (5′-cap), or at the 3′-terminus (3′-cap), orcan be present on both termini. Cap structures include, for example,inverted deoxy abasic caps.

Certain Pharmaceutical Compositions

Provided herein are pharmaceutical compositions comprisingoligonucleotides. In certain embodiments, a pharmaceutical compositionprovided herein comprises a compound comprising a modifiedoligonucleotide consisting of 15 to 25 linked nucleosides and having anucleobase sequence complementary to miR-21. In certain embodiments, apharmaceutical composition provided herein comprises a compoundconsisting of a modified oligonucleotide consisting of 8 to 30 linkednucleosides and having a nucleobase sequence complementary to miR-21. Incertain embodiments, a pharmaceutical composition provided hereincomprises a compound comprising a modified oligonucleotide consisting of12 to 25 linked nucleosides and having a nucleobase sequencecomplementary to miR-21.

Suitable administration routes include, but are not limited to, oral,rectal, transmucosal, intestinal, enteral, topical, suppository, throughinhalation, intrathecal, intracardiac, intraventricular,intraperitoneal, intranasal, intraocular, intratumoral, and parenteral(e.g., intravenous, intramuscular, intramedullary, and subcutaneous). Incertain embodiments, pharmaceutical intrathecals are administered toachieve local rather than systemic exposures. For example,pharmaceutical compositions may be injected directly in the area ofdesired effect (e.g., into the kidney).

In certain embodiments, a pharmaceutical composition is administered inthe form of a dosage unit (e.g., tablet, capsule, bolus, etc.). In someembodiments, a pharmaceutical compositions comprises a modifiedoligonucleotide at a dose within a range selected from 25 mg to 800 mg,25 mg to 700 mg, 25 mg to 600 mg, 25 mg to 500 mg, 25 mg to 400 mg, 25mg to 300 mg, 25 mg to 200 mg, 25 mg to 100 mg, 100 mg to 800 mg, 200 mgto 800 mg, 300 mg to 800 mg, 400 mg to 800 mg, 500 mg to 800 mg, 600 mgto 800 mg, 100 mg to 700 mg, 150 mg to 650 mg, 200 mg to 600 mg, 250 mgto 550 mg, 300 mg to 500 mg, 300 mg to 400 mg, and 400 mg to 600 mg. Incertain embodiments, such pharmaceutical compositions comprise amodified oligonucleotide in a dose selected from 25 mg, 30 mg, 35 mg, 40mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90mg, 95 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135mg, 140 mg, 145 mg, 150 mg, 155 mg, 160 mg, 165 mg, 170 mg, 175 mg, 180mg, 185 mg, 190 mg, 195 mg, 200 mg, 205 mg, 210 mg, 215 mg, 220 mg, 225mg, 230 mg, 235 mg, 240 mg, 245 mg, 250 mg, 255 mg, 260 mg, 265 mg, 270mg, 270 mg, 280 mg, 285 mg, 290 mg, 295 mg, 300 mg, 305 mg, 310 mg, 315mg, 320 mg, 325 mg, 330 mg, 335 mg, 340 mg, 345 mg, 350 mg, 355 mg, 360mg, 365 mg, 370 mg, 375 mg, 380 mg, 385 mg, 390 mg, 395 mg, 400 mg, 405mg, 410 mg, 415 mg, 420 mg, 425 mg, 430 mg, 435 mg, 440 mg, 445 mg, 450mg, 455 mg, 460 mg, 465 mg, 470 mg, 475 mg, 480 mg, 485 mg, 490 mg, 495mg, 500 mg, 505 mg, 510 mg, 515 mg, 520 mg, 525 mg, 530 mg, 535 mg, 540mg, 545 mg, 550 mg, 555 mg, 560 mg, 565 mg, 570 mg, 575 mg, 580 mg, 585mg, 590 mg, 595 mg, 600 mg, 605 mg, 610 mg, 615 mg, 620 mg, 625 mg, 630mg, 635 mg, 640 mg, 645 mg, 650 mg, 655 mg, 660 mg, 665 mg, 670 mg, 675mg, 680 mg, 685 mg, 690 mg, 695 mg, 700 mg, 705 mg, 710 mg, 715 mg, 720mg, 725 mg, 730 mg, 735 mg, 740 mg, 745 mg, 750 mg, 755 mg, 760 mg, 765mg, 770 mg, 775 mg, 780 mg, 785 mg, 790 mg, 795 mg, and 800 mg. Incertain such embodiments, a pharmaceutical composition of the comprisesa dose of modified oligonucleotide selected from 25 mg, 50 mg, 75 mg,100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 500 mg, 600 mg,700 mg, and 800 mg.

In certain embodiments, a pharmaceutical agent is sterile lyophilizedmodified oligonucleotide that is reconstituted with a suitable diluent,e.g., sterile water for injection or sterile saline for injection. Thereconstituted product is administered as a subcutaneous injection or asan intravenous infusion after dilution into saline. The lyophilized drugproduct consists of a modified oligonucleotide which has been preparedin water for injection, or in saline for injection, adjusted to pH7.0-9.0 with acid or base during preparation, and then lyophilized. Thelyophilized modified oligonucleotide may be 25-800 mg of anoligonucleotide. It is understood that this encompasses 25, 50, 75, 100,125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 425, 450, 475,500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, and 800 mgof modified lyophilized oligonucleotide. Further, in some embodiments,the lyophilized modified oligonucleotide is an amount of anoligonucleotide within a range selected from 25 mg to 800 mg, 25 mg to700 mg, 25 mg to 600 mg, 25 mg to 500 mg, 25 mg to 400 mg, 25 mg to 300mg, 25 mg to 200 mg, 25 mg to 100 mg, 100 mg to 800 mg, 200 mg to 800mg, 300 mg to 800 mg, 400 mg to 800 mg, 500 mg to 800 mg, 600 mg to 800mg, 100 mg to 700 mg, 150 mg to 650 mg, 200 mg to 600 mg, 250 mg to 550mg, 300 mg to 500 mg, 300 mg to 400 mg, and 400 mg to 600 mg. Thelyophilized drug product may be packaged in a 2 mL Type I, clear glassvial (ammonium sulfate-treated), stoppered with a bromobutyl rubberclosure and sealed with an aluminum FLIP-OFF® overseal.

In certain embodiments, the pharmaceutical compositions provided hereinmay additionally contain other adjunct components conventionally foundin pharmaceutical compositions, at their art-established usage levels.Thus, for example, the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the oligonucleotide(s) of the formulation.

Lipid moieties have been used in nucleic acid therapies in a variety ofmethods. In one method, the nucleic acid is introduced into preformedliposomes or lipoplexes made of mixtures of cationic lipids and neutrallipids. In another method, DNA complexes with mono- or poly-cationiclipids are formed without the presence of a neutral lipid. In certainembodiments, a lipid moiety is selected to increase distribution of apharmaceutical agent to a particular cell or tissue. In certainembodiments, a lipid moiety is selected to increase distribution of apharmaceutical agent to fat tissue. In certain embodiments, a lipidmoiety is selected to increase distribution of a pharmaceutical agent tomuscle tissue.

In certain embodiments, INTRALIPID is used to prepare a pharmaceuticalcomposition comprising an oligonucleotide. Intralipid is fat emulsionprepared for intravenous administration. It is made up of 10% soybeanoil, 1.2% egg yolk phospholipids, 2.25% glycerin, and water forinjection. In addition, sodium hydroxide has been added to adjust the pHso that the final product pH range is 6 to 8.9.

In certain embodiments, a pharmaceutical composition provided hereincomprise a polyamine compound or a lipid moiety complexed with a nucleicacid. In certain embodiments, such preparations comprise one or morecompounds each individually having a structure defined by formula (Z) ora pharmaceutically acceptable salt thereof,

wherein each X^(a) and X^(b), for each occurrence, is independently C₁₋₆alkylene; n is 0, 1, 2, 3, 4, or 5; each R is independently H, whereinat least n+2 of the R moieties in at least about 80% of the molecules ofthe compound of formula (Z) in the preparation are not H; m is 1, 2, 3or 4; Y is O, NR², or S; R¹ is alkyl, alkenyl, or alkynyl; each of whichis optionally substituted with one or more substituents; and R² is H,alkyl, alkenyl, or alkynyl; each of which is optionally substituted eachof which is optionally substituted with one or more substituents;provided that, if n=0, then at least n+3 of the R moieties are not H.Such preparations are described in PCT publication WO/2008/042973, whichis herein incorporated by reference in its entirety for the disclosureof lipid preparations. Certain additional preparations are described inAkinc et al., Nature Biotechnology 26, 561-569 (1 May 2008), which isherein incorporated by reference in its entirety for the disclosure oflipid preparations.

In certain embodiments, pharmaceutical compositions provided hereincomprise one or more modified oligonucleotides and one or moreexcipients. In certain such embodiments, excipients are selected fromwater, salt solutions, alcohol, polyethylene glycols, gelatin, lactose,amylase, magnesium stearate, talc, silicic acid, viscous paraffin,hydroxymethylcellulose and polyvinylpyrrolidone.

In certain embodiments, a pharmaceutical composition provided herein isprepared using known techniques, including, but not limited to mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or tableting processes.

In certain embodiments, a pharmaceutical composition provided herein isa liquid (e.g., a suspension, elixir and/or solution). In certain ofsuch embodiments, a liquid pharmaceutical composition is prepared usingingredients known in the art, including, but not limited to, water,glycols, oils, alcohols, flavoring agents, preservatives, and coloringagents.

In certain embodiments, a pharmaceutical composition provided herein isa solid (e.g., a powder, tablet, and/or capsule). In certain of suchembodiments, a solid pharmaceutical composition comprising one or moreoligonucleotides is prepared using ingredients known in the art,including, but not limited to, starches, sugars, diluents, granulatingagents, lubricants, binders, and disintegrating agents.

In certain embodiments, a pharmaceutical composition provided herein isformulated as a depot preparation. Certain such depot preparations aretypically longer acting than non-depot preparations. In certainembodiments, such preparations are administered by implantation (forexample subcutaneously or intramuscularly) or by intramuscularinjection. In certain embodiments, depot preparations are prepared usingsuitable polymeric or hydrophobic materials (for example an emulsion inan acceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

In certain embodiments, a pharmaceutical composition provided hereincomprises a delivery system. Examples of delivery systems include, butare not limited to, liposomes and emulsions. Certain delivery systemsare useful for preparing certain pharmaceutical compositions includingthose comprising hydrophobic compounds. In certain embodiments, certainorganic solvents such as dimethylsulfoxide are used.

In certain embodiments, a pharmaceutical composition provided hereincomprises one or more tissue-specific delivery molecules designed todeliver the one or more pharmaceutical agents of the present inventionto specific tissues or cell types. For example, in certain embodiments,pharmaceutical compositions include liposomes coated with atissue-specific antibody.

In certain embodiments, a pharmaceutical composition provided hereincomprises a sustained-release system. A non-limiting example of such asustained-release system is a semi-permeable matrix of solid hydrophobicpolymers. In certain embodiments, sustained-release systems may,depending on their chemical nature, release pharmaceutical agents over aperiod of hours, days, weeks or months.

In certain embodiments, a pharmaceutical composition is prepared foradministration by injection (e.g., intravenous, subcutaneous,intramuscular, etc.). In certain of such embodiments, a pharmaceuticalcomposition comprises a carrier and is formulated in aqueous solution,such as water or physiologically compatible buffers such as Hanks'ssolution, Ringer's solution, or physiological saline buffer. In certainembodiments, other ingredients are included (e.g., ingredients that aidin solubility or serve as preservatives). In certain embodiments,injectable suspensions are prepared using appropriate liquid carriers,suspending agents and the like. Certain pharmaceutical compositions forinjection are presented in unit dosage form, e.g., in ampoules or inmulti-dose containers. Certain pharmaceutical compositions for injectionare suspensions, solutions or emulsions in oily or aqueous vehicles, andmay contain formulatory agents such as suspending, stabilizing and/ordispersing agents. Certain solvents suitable for use in pharmaceuticalcompositions for injection include, but are not limited to, lipophilicsolvents and fatty oils, such as sesame oil, synthetic fatty acidesters, such as ethyl oleate or triglycerides, and liposomes. Aqueousinjection suspensions may contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, such suspensions may also contain suitablestabilizers or agents that increase the solubility of the pharmaceuticalagents to allow for the preparation of highly concentrated solutions.

In certain embodiments, a pharmaceutical composition provided hereincomprises a modified oligonucleotide in a therapeutically effectiveamount. In certain embodiments, the therapeutically effective amount issufficient to prevent, alleviate or ameliorate symptoms of a disease orto prolong the survival of the subject being treated. Determination of atherapeutically effective amount is well within the capability of thoseskilled in the art.

In certain embodiments, one or more modified oligonucleotides providedherein is formulated as a prodrug. In certain embodiments, upon in vivoadministration, a prodrug is chemically converted to the biologically,pharmaceutically or therapeutically more active form of anoligonucleotide. In certain embodiments, prodrugs are useful becausethey are easier to administer than the corresponding active form. Forexample, in certain instances, a prodrug may be more bioavailable (e.g.,through oral administration) than is the corresponding active form. Incertain instances, a prodrug may have improved solubility compared tothe corresponding active form. In certain embodiments, prodrugs are lesswater soluble than the corresponding active form. In certain instances,such prodrugs possess superior transmittal across cell membranes, wherewater solubility is detrimental to mobility. In certain embodiments, aprodrug is an ester. In certain such embodiments, the ester ismetabolically hydrolyzed to carboxylic acid upon administration. Incertain instances the carboxylic acid containing compound is thecorresponding active form. In certain embodiments, a prodrug comprises ashort peptide (polyaminoacid) bound to an acid group. In certain of suchembodiments, the peptide is cleaved upon administration to form thecorresponding active form.

In certain embodiments, a prodrug is produced by modifying apharmaceutically active compound such that the active compound will beregenerated upon in vivo administration. The prodrug can be designed toalter the metabolic stability or the transport characteristics of adrug, to mask side effects or toxicity, to improve the flavor of a drugor to alter other characteristics or properties of a drug. By virtue ofknowledge of pharmacodynamic processes and drug metabolism in vivo,those of skill in this art, once a pharmaceutically active compound isknown, can design prodrugs of the compound (see, e.g., Nogrady (1985)Medicinal Chemistry A Biochemical Approach, Oxford University Press, NewYork, pages 388-392).

Certain Additional Therapies

Treatments for a disease associated with miR-21 may comprise more thanone therapy. As such, in certain embodiments provided herein are methodsfor treating a subject having or suspected of having a diseaseassociated with miR-21 comprising administering at least one therapy inaddition to administering a modified oligonucleotide having a nucleobasesequence complementary to the microRNA.

In certain embodiments, the at least one additional therapy comprises apharmaceutical agent.

In certain embodiments, pharmaceutical agents include anti-inflammatoryagents. In certain embodiments, an anti-inflammatory agent is asteroidal anti-inflammatory agent. In certain embodiments, a steroidanti-inflammatory agent is a corticosteroid. In certain embodiments, acorticosteroid is prednisone. In certain embodiments, ananti-inflammatory agent is a non-steroidal anti-inflammatory drugs. Incertain embodiments, a non-steroidal anti-inflammatory agent isibuprofen, a COX-I inhibitors, or a COX-2 inhibitors.

In certain embodiments, pharmaceutical agents include anti-diabeticagents. Antidiabetic agents include, but are not limited to, biguanides,glucosidase inhibitors, insulins, sulfonylureas, and thiazolidenediones.

In certain embodiments, pharmaceutical agents include, but are notlimited to, diuretics (e.g. sprionolactone, eplerenone, furosemide),inotropes (e.g. dobutamine, milrinone), digoxin, vasodilators,angiotensin II converting enzyme (ACE) inhibitors (e.g. are captopril,enalapril, lisinopril, benazepril, quinapril, fosinopril, and ramipril),angiotensin II receptor blockers (ARB) (e.g. candesartan, irbesartan,olmesartan, losartan, valsartan, telmisartan, eprosartan), calciumchannel blockers, isosorbide dinitrate, hydralazine, nitrates (e.g.isosorbide mononitrate, isosorbide dinitrate), hydralazine,beta-blockers (e.g. carvedilol, metoprolol), and natriuretic peptides(e.g. nesiritide). In certain embodiments, an ACE inhibitor is selectedfrom cilazapril, perindopril, and trandolapril.

In certain embodiments, an ACE inhibitor is administered at a dose of0.025 to 0.1 mg/kg body weight. In certain embodiments, an ACE inhibitoris administered at a dose of 0.125 to 1.0 mg/kg bodyweight. In certainembodiments, an ACE inhibitor is administered at a dose ranging from 1to 6 mg/m²/day. In certain embodiments, an ACE inhibitor is administeredat a dose ranging from 1 to 2 mg/m²/day. In certain embodiments, an ACEinhibitor is administered at a dose ranging from 2 to 4 mg/m²/day. Incertain embodiments, an ACE inhibitor is administered at a dose rangingfrom 0.5 to 1 mg/m²/day.

In certain embodiment, ramipril is administered at a dose ranging from 1to 6 mg/m²/day. In certain embodiments, ramipril is administered at adose ranging from 1 to 2 mg/m²/day. In certain embodiment, enalapril isadministered at a dose ranging from 2 to 4 mg/m²/day. In certainembodiment, lisinopril is administered at a dose ranging from 4 to 8mg/m²/day. In certain embodiment, benazepril is administered at a doseranging from 4 to 8 mg/m²/day. In certain embodiment, fosinopril isadministered at a dose ranging from 4 to 8 mg/m²/day. In certainembodiment, quinapril is administered at a dose ranging from 4 to 8mg/m²/day. In certain embodiment, cilazapril is administered at a doseranging from 1 to 2 mg/m²/day. In certain embodiment, perinpril isadministered at a dose ranging from 1 to 2 mg/m²/day. In certainembodiment, trandolapril is administered at a dose ranging from 0.5 to 1mg/m²/day.

In certain embodiments, an ARB is administered at a dose ranging from6.25 to 150 mg/m2/day. In certain embodiments, an ARB is administered ata dose of 6.25 mg/m²/day. In certain embodiments, an ARB is administeredat a dose of 10 mg/m²/day. In certain embodiments, an ARB isadministered at a dose of 12.5 mg/m²/day. In certain embodiments, an ARBis administered at a dose of 18.75 mg/m²/day. In certain embodiments, anARB is administered at a dose of 37.5 mg/m²/day. In certain embodiments,an ARB is administered at a dose of 50 mg/m²/day. In certainembodiments, an ARB is administered at a dose of 150 mg/m²/day.

In certain embodiments, losartan is administered at a dose of 12.5mg/m²/day. In certain embodiments, losartan is administered at a dose of12.5 mg/m²/day. In certain embodiments, candesartan is administered at adose of 6.25 mg/m²/day. In certain embodiments, irbestartan isadministered at a dose of 37.5 mg/m²/day. In certain embodiments,telmisartan is administered at a dose of 10 mg/m²/day. In certainembodiments, valsartan is administered at a dose of 18.75 mg/m²/day. Incertain embodiments, espresartan is administered at a dose of 150mg/m²/day.

In certain embodiments, a pharmaceutical agent is an aldosteroneantagonsist. In certain embodiments, an aldosterone antagonist isspironolactone. In certain embodiments, spironolactone is administeredat a dose ranging from 10 to 35 mg daily. In certain embodiments,spironolactone is administered at a dose of 25 mg daily.

In certain embodiments, pharmaceutical agents include heparinoids. Incertain embodiments, a heparinoid is pentosan polysulfate.

In certain embodiments, a pharmaceutical agent is a pharmaceutical agentthat blocks one or more responses to fibrogenic signals.

In certain embodiments, a pharmaceutical agent is an anti-connectivetissue growth factor therapy. In certain embodiments, an anti-CTGFtherapy is a monoclonal antibody against CTGF.

In certain embodiments, an additional therapy may be a pharmaceuticalagent that enhances the body's immune system, including low-dosecyclophosphamide, thymostimulin, vitamins and nutritional supplements(e.g., antioxidants, including vitamins A, C, E, beta-carotene, zinc,selenium, glutathione, coenzyme Q-10 and echinacea), and vaccines, e.g.,the immunostimulating complex (ISCOM), which comprises a vaccineformulation that combines a multimeric presentation of antigen and anadjuvant.

In certain embodiments, the additional therapy is selected to treat orameliorate a side effect of one or more pharmaceutical compositions ofthe present invention. Such side effects include, without limitation,injection site reactions, liver function test abnormalities, kidneyfunction abnormalities, liver toxicity, renal toxicity, central nervoussystem abnormalities, and myopathies. For example, increasedaminotransferase levels in serum may indicate liver toxicity or liverfunction abnormality. For example, increased bilirubin may indicateliver toxicity or liver function abnormality.

Further examples of additional pharmaceutical agents include, but arenot limited to, immunoglobulins, including, but not limited tointravenous immunoglobulin (IVIg); analgesics (e.g., acetaminophen);salicylates; antibiotics; antivirals; antifungal agents; adrenergicmodifiers; hormones (e.g., anabolic steroids, androgen, estrogen,calcitonin, progestin, somatostan, and thyroid hormones);immunomodulators; muscle relaxants; antihistamines; osteoporosis agents(e.g., biphosphonates, calcitonin, and estrogens); prostaglandins,antineoplastic agents; psychotherapeutic agents; sedatives; poison oakor poison sumac products; antibodies; and vaccines.

Certain Kits

The present invention also provides kits. In some embodiments, the kitscomprise one or more compounds of the invention comprising a modifiedoligonucleotide, wherein the nucleobase sequence of the oligonucleotideis complementary to the nucleobase sequence of miR-21. The compoundscomplementary to miR-21 can have any of the nucleoside patternsdescribed herein. In some embodiments, the compounds complementary tomiR-21 can be present within a vial. A plurality of vials, such as 10,can be present in, for example, dispensing packs. In some embodiments,the vial is manufactured so as to be accessible with a syringe. The kitcan also contain instructions for using the compounds complementary tomiR-21.

In some embodiments, the kits may be used for administration of thecompound complementary to miR-21 to a subject. In such instances, inaddition to compounds complementary to miR-21, the kit can furthercomprise one or more of the following: syringe, alcohol swab, cottonball, and/or gauze pad. In some embodiments, the compounds complementaryto miR-21 can be present in a pre-filled syringe (such as a single-dosesyringes with, for example, a 27 gauge, ½ inch needle with a needleguard), rather than in a vial. A plurality of pre-filled syringes, suchas 10, can be present in, for example, dispensing packs. The kit canalso contain instructions for administering the compounds complementaryto miR-21.

Certain Experimental Models

In certain embodiments, the present invention provides methods of usingand/or testing modified oligonucleotides of the present invention in anexperimental model. Those having skill in the art are able to select andmodify the protocols for such experimental models to evaluate apharmaceutical agent of the invention.

Generally, modified oligonucleotides are first tested in cultured cells.Suitable cell types include those that are related to the cell type towhich delivery of a modified oligonucleotide is desired in vivo. Forexample, suitable cell types for the study of the methods describedherein include primary or cultured cells.

In certain embodiments, the extent to which a modified oligonucleotideinterferes with the activity of miR-21 is assessed in cultured cells. Incertain embodiments, inhibition of microRNA activity may be assessed bymeasuring the levels of the microRNA. Alternatively, the level of apredicted or validated microRNA-regulated transcript may be measured. Aninhibition of microRNA activity may result in the increase in themiR-21-regulated transcript, and/or the protein encoded bymiR-21-regulated transcript. Further, in certain embodiments, certainphenotypic outcomes may be measured.

Several animal models are available to the skilled artisan for the studyof miR-21 in models of human disease. For example, inhibitors of miR-21may be studied in an experimental model of Alport Syndrome, for exampleCol4a3 knockout mice (Col4a3^(−/−) mice). The severity of the disease inthe mouse model depends upon the genetic background of the mousecarrying the Col4a3 mutation. For example, the onset and progression ofthe disease are generally more rapid on the 129X1/SvJ relative to theC57BL/6J background. Accordingly, the genetic background of theCol4a3^(−/−) mouse may be selected to vary the onset and progression ofdisease. Additional models include canine models of X-linked, autosomalrecessive or autosomal dominant Alport Syndrome. See, for example,Kashtan, Nephrol. Dial. Transplant, 2002, 17: 1359-1361.

Certain Quantitation Assays

The effects of antisense inhibition of miR-21 following theadministration of modified oligonucleotides may be assessed by a varietyof methods known in the art. In certain embodiments, these methods arebe used to quantitate microRNA levels in cells or tissues in vitro or invivo. In certain embodiments, changes in microRNA levels are measured bymicroarray analysis. In certain embodiments, changes in microRNA levelsare measured by one of several commercially available PCR assays, suchas the TaqMan® MicroRNA Assay (Applied Biosystems). In certainembodiments, antisense inhibition of miR-21 is assessed by measuring themRNA and/or protein level of a target of miR-21. Antisense inhibition ofmiR-21 generally results in the increase in the level of mRNA and/orprotein of a target of the microRNA.

Target Engagement Assay

Modulation of microRNA activity with an anti-miR or microRNA mimic maybe assessed by measuring target engagement. In certain embodiments,target engagement is measured by microarray profiling of mRNAs. Thesequences of the mRNAs that are modulated (either increased ordecreased) by the anti-miR or microRNA mimic are searched for microRNAseed sequences, to compare modulation of mRNAs that are targets of themicroRNA to modulation of mRNAs that are not targets of the microRNA. Inthis manner, the interaction of the anti-miR with miR-21, or miR-21mimic with its targets, can be evaluated. In the case of an anti-miR,mRNAs whose expression levels are increased are screened for the mRNAsequences that comprise a seed match to the microRNA to which theanti-miR is complementary.

EXAMPLES

The following examples are presented in order to more fully illustratesome embodiments of the invention. They should in no way be construed,however, as limiting the broad scope of the invention. Those of ordinaryskill in the art will readily adopt the underlying principles of thisdiscovery to design various compounds without departing from the spiritof the current invention.

Example 1: Anti-miR-21 in a Model of Alport Syndrome

Col4a3^(−/−) mice on the 129sv genetic background spontaneously developsevere kidney disease similar to human Alport Syndrome. As such,Col4a3^(−/−) mice are used as an experimental model of Alport Syndrome.

Modified oligonucleotides complementary to miR-21 (anti-miR-21compounds) were tested in the Col4a3^(−/−) model of Alport Syndrome.Wild-type mice were used as control mice.

The structure of the anti-miR-21 compound is5′-A_(E)C_(S)ATC_(S)AGTC_(S)TGAU_(S)AAGC_(S)TA_(E)-3′ (SEQ ID NO: 3),where nucleosides not followed by a subscript indicateβ-D-deoxyribonucleosides; nucleosides followed by a subscript “E”indicate 2′-MOE nucleosides; nucleosides followed by a subscript “S”indicate S-cEt nucleosides. Each internucleoside linkage is aphosphorothioate internucleoside linkage.

At 3 weeks of age, mice were genotyped to identify Col4a3−/− mice. From3 weeks of age to 9 weeks of age, sex-matched littermates of mice weretreated with anti-miR-21 or PBS. Anti-miR-21 was administeredsubcutaneously at a dose of 25 mg/kg, twice per week. Treatment groupswere: (1) wild-type mice, PBS administration, n=8; (2) Col4a3−/− mice,PBS administration, n=12; (3) Col4a3−/− mice, anti-miR-21administration, n=12. Wild type littermates of Col4a3−/− mice were usedas the wild-type control mice. An overnight urine sample (approximately16 hours) was collected weekly. Plasma and kidneys were harvested at theend of week 9. Fluid and tissue samples were analyzed to determinechanges in kidney function, kidney damage and glomerular sclerosis andinterstitial fibrosis.

Endpoints in blood or urine included measurement of blood urea nitrogen(BUN), albuminuria, albumin/creatinine ratio, glomerular filtrationrate. Histological analysis included evaluation of glomerular sclerosis,interstitial fibrosis, injury to the tubules, macrophage infiltration,and presence of myofibroblasts.

Blood urea nitrogen (BUN) was measured at week 9. Statisticalsignificance was calculated by the Mann Whitney test. As shown in FIG.1A, a statistically significant reduction in BUN was observed in animalstreated with anti-miR-21, relative to PBS-treated control animals at theend of the study. The reduced BUN was observed overall (FIG. 1A), aswell as in male mice only (approximately 90 mg/dL compared toapproximately 25 mg/dL in control male mice) and female mice only(approximately 70 mg/dL compared to approximately 25 mg/dL in controlfemale mice) not shown). The BUN in Col4a3+/+mice was approximately 12.5mg/kL (within normal limits; not shown). BUN is a blood marker of kidneyfunction. Higher BUN correlates with poorer kidney function. A reductionin BUN is an indicator of reduced kidney injury and damage and improvedfunction.

Albuminuria was assessed by measuring albumin in urine samples,collected over 16 hours at a frequency of once weekly, by ELISA and bynormalizing to urinary creatinine excretion. All analyses were performedat the same time at the end of the study. As shown in FIG. 1B, Col4a3−/−mice develop severe albuminuria. However, mice treated with anti-miR-21developed much less albuminuria as detected by a reduction in urinaryalbumin to creatinine ratio. This reduction was observed by week 7 andpersisted to week 9. Wild type littermates of Col4a3−/− mice exhibitedno albuminuria, as expected. Albuminuria is a sensitive measure ofglomerular and tubular damage. A reduction in albumin to creatinineratio indicates a reduction in glomerular and/or tubular disease.

Alport Syndrome is also characterized by progressive development ofglomerulosclerosis and significant interstitial kidney fibrosis thatoccurs as inappropriate glomerular leakage occurs. Accordingly,glomerulosclerosis was assessed by blinded scoring of glomeruli forsclerotic lesions (loss of capillary loop+fibrosis or hyalinosis).Thirty glomeruli were scored sequentially from each mouse by a blindedobserver. The score was from 0-4 where 0=normal; 1=<25% of theglomerulus affected by sclerosis; 2=25-50% of the glomerulus is affectedby sclerosis; 3=50-75% of the glomerulus is affected by sclerosis;4=75-100% of the glomerulus is affected by sclerosis. The proportion ofglomeruli with no disease was much higher in mice treated withanti-miR-21 and the proportion of glomeruli with moderately or severelyaffected glomeruli (score 2-4) was significantly higher in the micetreated with the PBS (FIG. 2). Glomeruli were also scored in wild typelittermates (WT) of Col43a−/− mice. Interstitial fibrosis was measuredmorphometrically in whole sagittal sections stained with Picrosirius redfrom PBS-treated and anti-miR-21 Col4a3−/− animals. As shown in FIG. 3A,a statistically significant reduction in interstitial fibrosis wasobserved in the anti-miR-21 treated Col4a3−/− mice. In addition,quantitative PCR for the transcripts for the major pathological matrixprotein Collagen Iα(1) (Col1a1) showed that the kidney tissue fromanti-miR-21 treated Col4a3−/− mice showed much less production of thispathological collagen (FIG. 3B).

Renal tissue injury was assessed in paraffin-embedded andparaformaldehyde (4%)-fixed tissue sections stained by the periodicacid-Schiff (PAS) reaction. Initially the kidney sections were rankedfor overall injury based on tubule and glomerular injury andinflammation. Damage was assessed based on a variety of factorsincluding tubule dilation, loss of brush border, cellular infiltration,glomerular inflammation, interstitial edema and cellular necrosis.Kidney sections were ranked in a blinded fashion for overall injury andgiven a kidney injury rank score. The kidney sections from Col4a3−/−mice showed a significantly lower kidney injury rank score, which isindicative of less kidney injury (FIG. 4A). To analyze this in moredetail, the glomeruli were assessed by a blinded observer for theproportion that had glomerular crescents. The crescent is aproliferation of cells within Bowman's capsule, is defined by ≥2 layersof cells within Bowman's space. The crescent is a well-establishedmarker of glomerular injury. In Col4a3−/− mice that received anti-miR-21the proportion of glomeruli with crescents was approximately 44%,whereas in mice that received the PBS control treatment, the proportionof glomeruli with crescents was approximately 19% (FIG. 4B). InCol4a3+/+ littermates, the proportion of glomeruli with crescents wasless than 5% (not shown). The tubules of the nephrons of the kidney arealso a site for damage. The tubule damage was assessed by overlaying agrid over sequential images covering the whole sagittal section of eachkidney. In a blinded fashion, damage of the tubules was assessed in eachsquare of the grid. Tubular damage was assessed based on the presence oftubule dilation/flattening, loss of brush border, cellular infiltration,and cellular necrosis. The presence of these features results in apositive score for that square on the grid. An overall score is appliedto each image which is the % of squares that has tubule damage. This isaveraged for all the images from that kidney. The average score for eachkidney is then subjected to statistical analysis. As is shown, thetubule injury score was significantly lower in the Col4a3−/− micetreated with anti-miR-21, relative to the Col4a3−/− receiving PBS (FIG.4C). The tubule injury score in Col4a3+/+ littermates was less than 10%(not shown).

Additional histological analysis of kidney samples was performed toevaluate macrophage infiltration, endothelial stability, and themyofibroblast deposition. As judged by F4/80 staining, macrophageinfiltration was reduced in anti-miR-21 treated Col4a3−/− mice comparedto PBS-treated Col4a3−/− control mice (FIG. 5A). Immunocytochemicalstaining for CD31 demonstrated an improvement in endothelial stabilityin anti-miR-21 treated Col4a3−/− mice compared to PBS-treated Col4a3−/−control mice (not shown). Detection of alpha-SMA revealed a reduction inmyofibroblast deposition in anti-miR-21 treated Col4a3−/− mice comparedto PBS-treated Col4a3−/− control mice (FIG. 5B). In Col4a3+/+ mice,alpha-SMA staining was approximately 5% (not shown).

Reactive oxygen species (ROS) are a byproduct of normal cellularmetabolism. During cellular stress, excess ROS can cause lipidperoxidation of cell and organelle membranes, resulting in disruption ofthe structural integrity and capacity for cell transport and energyproduction. In the kidney, ROS produced during cellular stress can causerenal injury. To assess whether the generation of ROS was reducedfollowing inhibition of miR-21 in Col4a3−/− mice, urinary hydrogenperoxide levels were measured in anti-miR-21 and PBS-treated mice.Urinary hydrogen peroxide levels were significantly reduced in mice thatreceived anti-miR-21 (FIG. 6A). In Col4a3+/+ mice, urinary hydrogenperoxide levels were less than 5 μM (not shown). Further,immunocytochemical staining of kidney tissue with dihydroethidium (DHE),which is a measure of ROS, demonstrated a reduction in ROS in the kidneytissue of anti-miR-21 treated Col4a3−/− mice compared to PBS-treatedcontrol mice (FIG. 6B). In Col4a3+/+ mice, less than 10% DHE stainingwas observed (not shown). These data demonstrate reduction of ROS inboth the urine and kidney tissue in Col4a3−/− mice treated withanti-miR-21. Accordingly, one mechanism by which anti-miR-21 may reducekidney injury may include a reduction in the generation of reactiveoxygen species.

Immunoblotting of protein in the kidneys of Col4a3−/− mice treated withanti-miR-21 revealed an increase in the amount of MPV17L protein in thekidney, relative to Col4a3−/− mice. MPV17L is a mitochondrial innermembrane protein that is implicated in the metabolism of reactive oxygenspecies, and protects against oxidative stress. Accordingly, the reducedgeneration of ROS following anti-miR-21 treatment may occur at least inpart due to increased MPV17L levels. To further explore the mechanisticeffects of anti-miR-21, PPAR-alpha protein was measured byimmunoblotting of the kidneys of Col4a3−/− mice treated with PBS oranti-miR-21 treatment. Anti-miR-21 treatment increased PPAR-alphaprotein, suggesting a stimulation of metabolic pathways.

Podocytes are highly specialized epithelial cells that are an essentialcomponent of the glomerular filtration barrier. Podocyte loss can leadto proteinuria, and in some disease states to glomerularsclerosis. Toevaluate whether podocyte number was affected by inhibition of miR-21 inCol4a3−/− mice, podocyte number was measured in anti-miR-21 andPBS-treated mice. Podocyte number was significantly increased inCol4a3−/− mice that received anti-miR-21, relative to PBS-treated mice,and was comparable to the podocyte number observed in wild typelittermates of Col4a3−/− mice (FIG. 7). Accordingly, one mechanism bywhich anti-miR-21 may reduce kidney injury in a model of Alport Syndromeis by preventing or reducing podocyte loss.

A similar study was conducted using the following anti-miR-21 compounds:

anti-miR-21 compound #1 (above): (SEQ ID NO: 3)5′-A_(E)C_(S)ATC_(S)AGTC_(S)TGAU_(S)AAGC_(S)TA_(E)-3′anti-miR-21 compound #2: (SEQ ID NO: 3)5′-A_(E)C_(S)ATC_(S)A_(S)GTC_(S)U_(S)GAU_(S)A_(S)AGC_(S)U_(S)A_(E)-3′;anti-miR-21 compound #3: (SEQ ID NO: 4)5′-^(Me)C_(E)A_(S)A_(S)T_(E)C_(S)U_(S)A_(E)A_(E)U_(S)A_(S)A_(E)G_(E)C_(S)T_(E)A_(S)-3′;and anti-miR-21 compound #4: (SEQ ID NO: 3)5′-A_(E)C_(S)A_(E)T_(E)C_(S)A_(E)G_(E)T_(E)C_(S)TGAU_(S)AAGC_(S)U_(S)A_(S)-3′;where nucleosides not followed by a subscript indicateβ-D-deoxyribonucleosides; nucleosides followed by a subscript “E”indicate 2′-MOE nucleosides; nucleosides followed by a subscript “S”indicate S-cEt nucleosides; and a superscript “Me” indicates a 5-methylgroup on the base of the nucleoside. Each internucleoside linkage is aphosphorothioate internucleoside linkage.

Each compound was administered to three-week old Col4a3−/− mice at adose of 25 mg/kg, twice weekly, for nine weeks. Control groups includedCol4a3−/− mice treated with PBS, and wild-type littermates of Col4a3−/−mice. Each treatment group contained 10 to 12 mice. For compounds #1, 2,and 4, endpoints were evaluated as described above and included BUN,urinary albumin to creatinine ratio, kidney injury (PAS staining),glomerulosclerosis, and proportion of glomeruli with crescents. Forcompound #3, endpoints included BUN, urinary albumin to creatinineratio, and collagen gene expression (as a measure of fibrosis),evaluated as described above.

Consistent with the results described above, anti-miR-21 compound #1improved all endpoints evaluated. The efficacy of both anti-miR-21compounds #2 was similar to that of compound #1, with improvementsobserved in BUN, urinary albumin to creatinine ratio, kidney injury,extent of glomerulosclerosis, and percentage of glomeruli withcrescents. The efficacy of compound #3 was similar to that of compound#1, with improvements in BUN, urinary albumin to creatinine ratio, andCol1a1 expression. Anti-miR-21 compound #4, while less efficacious thancompounds the other compounds tested, still resulted in improvements inBUN, kidney injury, extent of glomerulosclerosis, and percentage ofglomeruli with crescents.

Taken together, these data illustrate that in a model of AlportSyndrome, anti-miR-21 treatment attenuated the loss of kidney functionand development of albuminuria. Glomerulosclerosis and interstitialfibrosis were markedly attenuated and proximal tubules were preserved.As anti-miR-21 prevents progressive loss of kidney function in theCol4a3−/− mouse, and attenuates both glomerular and tubulo-interstitialdisease, anti-miR-21 is a therapeutic agent for human Alport Syndrome.

Example 2: Elevation of miR-21 in a Model of Alport Syndrome

To evaluate the dysregulation of miR-21 in an experimental model ofAlport Syndrome, miR-21 levels were measured in kidney tissue harvestedfrom mice. RNA was isolated from whole kidney and miR-21 was measured byquantitative PCR. In Col4a3−/− mice, miR-21 levels were elevatedapproximately three-fold relative to miR-21 levels in wild-type mice.

Accordingly, a subject receiving treatment for Alport Syndrome may beidentified as having elevated miR-21 in kidney biopsy material, urine,or blood, prior to administration of the treatment.

Example 3: Survival Studies in a Model of Alport Syndrome

Wild-type mice generally live for 2 to 3 years (or 730 to 1095 days). InCol4a3−/− mice on a 129X1/SvJ background, end-stage renal failure canoccur as early as 2 months of age. In Col4a3−/− on a C57BL/6Jbackground, end-stage renal failure can occur as early as 6 months ofage. Regardless of the background, the lifespan of Col4a3−/− mice issignificantly shorter than that of wild-type mice. As such, Col4a3−/−mice, on any genetic background, can be serve as a model for end-stagerenal failure in Alport Syndrome and can be used to evaluate the effectsof candidate therapeutic agents on life expectancy.

Mice are genotyped to identify Col4a3−/− mice. Anti-miR-21 isadministered subcutaneously at a dose of ranging from 10 to 25 mg/kg,once or twice per week for up to one year. PBS may be administered as acontrol treatment. Overnight urine samples (approximately 16 hours) arecollected on a weekly or monthly schedule throughout the study. Age ofeach mouse at death is recorded. Plasma and kidneys are collected atdeath or at the end of the study. Fluid and tissue samples are analyzedto determine changes in kidney function, glomerularsclerosis, andfibrosis.

Fluid and tissue samples are analyzed to determine changes in kidneyfunction, kidney damage and glomerular sclerosis and interstitialfibrosis. Endpoints in blood or urine include measurement of blood ureanitrogen (BUN), albuminuria, albumin/creatinine ratio, glomerularfiltration rate. Histological analysis includes evaluation of glomerularsclerosis, interstitial fibrosis, injury to the tubules, macrophageinfiltration, and presence of myofibroblasts.

Delay in the onset of end-stage renal failure and increased lifeexpectancy in anti-miR-21 treated mice, relative to PBS-treated controlmice, is observed, suggesting that anti-miR-21 is a therapeutic agentthat can increase the life expectancy of subjects with Alport Syndrome.

Anti-miR-21 Increases Survival in a Model of Alport Syndrome-Single DoseStudy

To evaluate the effects of anti-miR-21 on survival in an experimentalmodel of Alport Syndrome, anti-miR-21 compound was administered toCol4a3−/− mice.

The structure of the anti-miR-21 compound is5′-A_(E)C_(S)ATC_(S)AGTC_(S)TGAU_(S)AAGC_(S)TA_(E)-3′ (SEQ ID NO: 3),where nucleosides not followed by a subscript indicateβ-D-deoxyribonucleosides; nucleosides followed by a subscript “E”indicate 2′-MOE nucleosides; nucleosides followed by a subscript “S”indicate S-cEt nucleosides. Each internucleoside linkage is aphosphorothioate internucleoside linkage.

Col4a3+/1 mice (heterozygotes) on a 129X1/SvJ background were crossed togenerate Col4a3−/− mice. At 3 weeks of age, mice were genotyped toidentify Col4a3−/− mice. Treatment groups were: (1) Col4a3+/+ mice(wild-type littermates), PBS administration, twice weekly, n=12; (2)Col4a3−/− mice, PBS administration, twice weekly, n=12; (3) Col4a3−/−mice, 25 mg/kg anti-miR-21 administration subcutaneously, twice weekly,n=12. Treatments were administered twice weekly, from week 3 throughweek 16. Animal weights were measured weekly, and lifespan was recorded.

As expected, Col4a3−/− mice experienced weight loss beginning at around9 weeks of age, and death occurred between 9 and 11 weeks of age. Asshown in FIG. 8A, anti-miR-21 increased peak body weight andsignificantly delayed weight loss (p<0.01). As shown in FIG. 8B,anti-miR-21 significantly increased lifespan (p<0.001). Thus, treatmentwith anti-miR-21 not only delayed the weight loss, but importantlyimproved survival of Col4a3−/− mice.

Anti-miR-21 Increases Survival in a Model of Alport Syndrome-DoseResponse Study

To evaluate the dose-responsive effects of anti-miR-21 on survival in anexperimental model of Alport Syndrome, several doses of anti-miR-21compound were administered to Col4a3−/− mice.

The structure of the anti-miR-21 compound is5′-A_(E)C_(S)ATC_(S)AGTC_(S)TGAU_(S)AAGC_(S)TA_(E)-3′ (SEQ ID NO: 3),where nucleosides not followed by a subscript indicateβ-D-deoxyribonucleosides; nucleosides followed by a subscript “E”indicate 2′-MOE nucleosides; nucleosides followed by a subscript “S”indicate S-cEt nucleosides. Each internucleoside linkage is aphosphorothioate internucleoside linkage.

At 3 weeks of age, mice were genotyped to identify Col4a3−/− mice.Treatment groups were:

(1) Col4a3−/− mice, PBS administration once weekly, n=13;

(2) Col4a3−/− mice, 12.5 mg/kg anti-miR-21 administration, once weekly,n=12;

(3) Col4a3−/− mice, 25 mg/kg anti-miR-21 administration, once weekly,n=13;

(4) Col4a3−/− mice, 50 mg/kg anti-miR-21 administration, once weekly,n=12;

(5) Col4a3−/− mice, 25 mg/kg anti-miR-21 administration, twice weekly,n=12;

Treatments were administered starting on day 24. Animal weights weremeasured weekly, and lifespan was recorded. At week 7, blood wascollected for measurement of BUN.

As shown in FIG. 9A, a reduction in BUN was observed in animals treatedwith anti-miR-21, relative to PBS-treated control animals. Although areduction in BUN was observed, it was not strongly dose-responsive,perhaps due to the fact that the disease was more severe in theCol4a3−/− mice used for this experiment (the mice were obtained from adifferent vendor than the Col4a3−/− mice described in the previousexamples). The observed reduction in BUN is an indicator of reducedkidney injury and damage and improved function.

As shown in FIG. 9B, treatment with anti-miR-21 increased the lifespanof Col4a3−/− mice in a dose responsive manner. The increased lifespanwas observed for both twice weekly and once weekly treatments. Themedian survival was as follows: PBS, 62 days; 12.5 mg/kg anti-miR-21once weekly (QW), 72.5 days; 25 mg/kg anti-miR-21 once weekly (QW), 77days; 50 mg/kg anti-miR-21 once weekly (QW), 89 days; 25 mg/kganti-miR-21 twice weekly (BIW), 82.5 days.

Delay in the onset of kidney dysfunction and increased life expectancyin anti-miR-21 treated mice, relative to PBS-treated control mice, wasobserved, suggesting that anti-miR-21 is a therapeutic agent that canincrease the life expectancy of subjects with Alport Syndrome.

Example 4: Anti-miR Distribution in the Kidney of Col4a3−/− Mice

Oligonucleotides, including anti-miR compounds, are known to distributeto several cell types within the kidney. As reported by Chau et al., SciTransl Med., 2012, 121ra18, following administration of a Cy3-labeledanti-miR to either normal mice or mice subjected to kidney injury(unilateral ureteral obstruction, a model of interstitial fibrosis), thegreatest fluorescence intensity in the kidney was in proximal tubuleepithelium. The endothelium, pericytes, myofibroblasts, and macrophagesalso all contained detectable amounts of Cy3-labeled anti-miR. However,the glomerulus, in particular podocytes, did not appear to take upsignificant amounts of anti-miR consistent with the known distributionof chemically modified oligonucleotides (Masarjian et al.,Oligonucleotides, 2004, 14, 299-310).

To investigate the distribution of anti-miR in a mouse model of AlportSyndrome, Cy3-labeled anti-miR compound was administered to twodifferent groups of Col4a3−/− mice, one at 6 weeks of age (n=3) and oneat 8 weeks of age (n=4) and to one group of wild type mice at 8 weeks ofage (n=3). Two days following administration of the anti-miR compound,animals were sacrificed and kidneys were harvested and processed forhistological analysis.

Sections of kidney tissue were co-labeled with antibodies specific toseveral different cellular markers to identify anti-miR uptake inparticular cell types Staining was performed for alpha-SMA (amyofibroblast marker), PDGFR-beta (a pericyte/myofibroblast marker),CD31 (an endothelial cell marker), F4/80 (a macrophage marker), and GP38(a podocyte marker). As expected, anti-miR compound was taken up intothe proximal tubule epithelium, pericytes, myofibroblasts, andmacrophages. In contrast to previous observations in normal mice andmice with interstitial fibrosis, in the Col4a3−/− mice anti-miR wastaken up into the glomerulus, including into podocytes.

As described herein, the efficacy observed following anti-miR-21administration in an experimental model of Alport Syndrome isaccompanied by improvements not only in interstitial fibrosissurrounding tubules but also fibrosis in the glomeruli (known asglomerulosclerosis). These data suggest that those improvements may bedirectly related to anti-miR-21 effects in the glomeruli, in addition toor instead of feedback from an improved tubule structure and function.

1-30. (canceled)
 31. A method of treating Alport Syndrome comprisingadministering to a subject having or suspected of having AlportSyndrome: (i) a pharmaceutical composition comprising a therapeuticallyeffective amount of a modified oligonucleotide consisting of 19 linkednucleosides and having the structure5′-A_(E)C_(S)ATC_(S)AGTC_(S)TGAU_(S)AAGC_(S)TA_(E)-3′ (SEQ ID NO: 3),where nucleosides not followed by a subscript areβ-D-deoxyribonucleosides; nucleosides followed by a subscript “E” are2′-MOE nucleosides; nucleosides followed by a subscript “S” are S-cEtnucleosides, and each internucleoside linkage is a phosphorothioateinternucleoside linkage; and (ii) at least one additional therapyselected from an angiotensin II converting enzyme (ACE) inhibitor, anangiotensin II receptor blocker (ARB), an anti-hypertensive agent, avitamin D analog, an oral phosphate binder, dialysis, and kidneytransplant.
 32. The method of claim 31, wherein the subject has beendiagnosed as having Alport Syndrome prior to administering the modifiedoligonucleotide and the at least one additional therapy.
 33. The methodof claim 31, wherein the administering: a) improves kidney function; b)delays the onset of end stage renal disease; c) delays time to dialysis;d) delays time to renal transplant; and/or e) improves life expectancy.34. The method of claim 31, wherein the administering: a) reduceshematuria; b) delays the onset of hematuria; c) reduces proteinuria; d)delays the onset of proteinuria; e) reduces kidney fibrosis; f) slowsfurther progression of fibrosis; and/or g) halts further progression offibrosis.
 35. The method of claim 31, wherein the subject has a mutationin the gene encoding the alpha 3 chain of type IV collagen, the alpha 4chain of type IV collagen, or the alpha 5 chain of type IV collagen. 36.The method of claim 31, wherein the subject is identified as havinghematuria, and/or proteinuria.
 37. The method of claim 31, wherein thesubject has reduced kidney function.
 38. The method of claim 31, whereinthe administering improves one or more markers of kidney function in thesubject, selected from: a) reduced blood urea nitrogen in the subject;b) reduced creatinine in the blood of the subject; c) improvedcreatinine clearance in the subject; d) reduced proteinuria in thesubject; e) reduced albumin:creatinine ratio in the subject; f) improvedglomerular filtration rate in the subject; g) reduced cystatin C in theblood of the subject; h) reduced β-trace protein (BTP) in the blood ofthe subject; i) reduced 2-microglobulin (B2M) in the blood of a subject;j) reduced NAG protein in the urine of the subject; k) reduced NGALprotein in the urine of the subject; l) reduced KIM-1 protein in theurine of the subject; m) reduced IL-18 protein in the urine of thesubject; n) reduced monocyte chemoattractant protein (MCP1) levels inthe urine of the subject; o) reduced connective tissue growth factor(CTGF) levels in the urine of the subject; p) reduced collagen IVfragments in the urine of the subject; q) reduced collagen III fragmentsin the urine of the subject; and/or r) reduced podocyte protein levelsin the urine of the subject, wherein the podocyte protein is selectedfrom nephrin and podocin.
 39. The method of claim 34, wherein theproteinuria is albuminuria.
 40. The method of claim 39, wherein thealbuminuria is high normal albuminuria, microalbuminuria, ormacroalbuminuria.
 41. The method of claim 31, wherein the AlportSyndrome is the X-linked form of Alport Syndrome.
 42. The method ofclaim 31, wherein the Alport Syndrome is the autosomal form of AlportSyndrome.
 43. The method of claim 31, wherein the method comprisesadministering an angiotensin II converting enzyme (ACE) inhibitorselected from captopril, enalapril, lisinopril, benazepril, quinapril,fosinopril, and ramipril.
 44. The method of claim 31, wherein the methodcomprises administering an angiotensin II receptor blocker (ARB)selected from candesartan, irbesartan, olmesartan, losartan, valsartan,telmisartan, and eprosartan.
 45. The method of claim 31, wherein themodified oligonucleotide is administered at a dose of 110 mg.
 46. Themethod of claim 43, wherein the modified oligonucleotide is administeredat a dose of 110 mg.
 47. The method of claim 44, wherein the modifiedoligonucleotide is administered at a dose of 110 mg.
 48. A method oftreating Alport Syndrome comprising administering to a subject having orsuspected of having Alport Syndrome a pharmaceutical compositioncomprising a therapeutically effective amount of a modifiedoligonucleotide consisting of 19 linked nucleosides and having thestructure 5′-A_(E)C_(S)ATC_(S)AGTC_(S)TGAU_(S)AAGC_(S)TA_(E)-3′ (SEQ IDNO: 3), wherein nucleosides not followed by a subscript areβ-D-deoxyribonucleosides, nucleosides followed by a subscript “E” are2′-O-methoxyethyl (2′MOE) nucleosides, nucleosides followed by asubscript “S” are S-cEt nucleosides, and each internucleoside linkage isa phosphorothioate internucleoside linkage; and wherein the modifiedoligonucleotide is administered at a dose of 110 mg.
 49. The method ofclaim 48, wherein the subject has been diagnosed as having AlportSyndrome prior to administering the pharmaceutical composition.
 50. Themethod of claim 48, wherein the Alport Syndrome is the X-linked form ofAlport Syndrome or the autosomal form of Alport Syndrome. 51-60.(canceled)